Compositions and methods for detecting or eliminating senescent cells to diagnose or treat disease

ABSTRACT

Disclosed are agents (e.g., peptides, polypeptides, proteins, small molecules, antibodies, and antibody fragments that target senescent cells) and methods of their use for imaging senescent cells in vivo and for treating or preventing cancer, age-related disease, tobacco-related disease, or other diseases and disorders related to or caused by cellular senescence in a mammal. The methods include administering one or more of the agents of the invention to a mammal, e.g., a human. The agents, which specifically bind to senescent cells, can be labeled with a radioactive label or a therapeutic label, e.g., a cytotoxic agent.

FIELD OF THE INVENTION

This invention relates to compositions and methods for the detection andtreatment of cancer, age-related diseases, tobacco-related diseases, andother diseases and disorders related to or caused by cellularsenescence.

BACKGROUND OF THE INVENTION

The definition of cellular senescence has undergone some revision sincethe phenomenon was described by Leonard Hayflick as cessation ofreplication of cultured human cells after a finite number of populationdoublings (Hayflick et al., Exp. Cell Res. 37:585-621, 1961). Senescentcells remain metabolically active but do not divide and resist apoptosisfor long periods of time (Goldstein, Science 249:1129-1133, 1990).Cellular senescence is characterized by growth cycle arrest in the G1phase, absence of S phase, and lifespan control by multiple dominantgenes (Stanulis-Praeger, Mech. Ageing Dev. 38:1-48, 1987).

Cellular senescence differs from quiescence and terminal differentiationin several important aspects. Senescent cells have characteristicmorphological changes such as enlargement, flattening, and increasedgranularity (Dimri et al., Proc. Nat. Acad. Sci. USA 92:9363-9367,1995). Senescent cells do not divide even if stimulated by mitogens(Campisi, Trends Cell Biol. 11:S27-S31, 2001). Senescence involvesactivation of p53 and/or Rb and their regulators such as p16^(INK4a),p21, and ARF. Except when p53 or Rb is inactivated, senescence isirreversible. Senescent cells express increased levels of plasminogenactivator inhibitor (PAI) and exhibit staining for β-galactosidaseactivity at pH 6 (Sharpless et al., J. Clin. Invest. 113:160-168, 2004).Irreversible G1 arrest is mediated by inactivation of cyclin dependentkinase (CdK) complexes which phosphorylate Rb. P21 accumulates in agingcells and inhibits CdK4-CdK6. P16 inhibits CdK4-CdK6 and accumulatesproportionally with β-galactosidase activity and cell volume (Stein etal., Mol. Cell. Biol. 19:2109-2117, 1999). P21 is expressed duringinitiation of senescence but need not persist; p16 expression helpsmaintain senescence once initiated.

Replicative senescence, the type of senescence originally observed byHayflick, is related to the progressive shortening of telomeres witheach cell division. Senescence is induced when certain chromosomaltelomeres reach a critical length (Mathon and Lloyd, Nat. Rev. Cancer3:203-213, 2001; Martins, U.M. Exp Cell Res. 256:291-299, 2000).Senescence can be abrogated by the expression of telomerase whichlengthens telomeres; human fibroblasts undergo replication indefinitelywhen transfected to express telomerase. Most cancers express telomerasein order to maintain telomere length and replicate indefinitely. Theminority of cancers that do not express telomerase have alternativelengthening of telomere (ALT) mechanisms.

Indirect evidence suggests some relationship between replicativesenescence and aging. Cultured cells from old donors exhibit senescenceafter fewer population doublings than cells from young donors (Martin etal., Lab. Invest. 23:86-92, 1970; Schneider et al., Proc. Nat. Acad.Sci. USA 73:3584-3588, 1976). Cells from short-lived species senesceafter fewer population doublings than cells from long-lived species(Rohme, D., Proc. Nat. Acad. Sci. USA 78:5009-3320, 1981). Culturedcells from donors with hereditary premature aging syndromes such asWerner's syndrome show senescence after fewer replications than cellsfrom age-matched controls (Goldstein, Genetics of Aging, 171-224, 1978;Martin, Genetic Effects on Aging, 5-39, 1990). Whether replicativesenescence actually contributes to aging or age-related symptoms in vivois questionable on the basis of theoretical estimates of the number ofcell divisions that occur in vivo and the absence of strong empiricalevidence.

There are, however, other pathways to senescence than replication.Collectively, these are often referred to as stress-induced prematuresenescence (SIPS). Oxidative stress can shorten telomeres (vonZglinicki, Trends Biochem. Sci. 27:339-344, 2002) and hyperoxia has beenshown to induce senescence. Gamma irradiation of human fibroblasts inearly to mid G1 phase causes senescence in a p53-dependent manner (DiLeonardo et al., Genes Dev. 8:2540-2551, 1994). Ultraviolet radiationalso induces cellular senescence. Other agents that can induce cellularsenescence include hydrogen peroxide (Krtolica et al., Proc. Nat. Acad.Sci. USA 98:12072-12077, 2001), sodium butyrate, 5-azacytadine, andtransfection with the Ras oncogene (Tominaga, Mech. Ageing Dev.123(8):927-936, 2002). Chemotherapeutic agents including doxorubicin,cisplatin, and a host of others have been shown to induce senescence incancer cells (Roninson, Cancer Res. 63:2705-2715, 2003).5-bromodeoxyuridine treatment results in cellular senescence in bothnormal and malignant cells (Michishita et al., J. Biochem.126:1052-1059, 1999). Generally speaking, agents that damage DNA cancause cellular senescence. The existence of cellular senescence in vivohas been demonstrated. In a study by Dimri et al., published in 1995,senescent fibroblasts were shown to exhibit staining for β-galactosidaseactivity at pH 6. These cells failed to incorporate tritiated thymidineand retained β-galactosidase activity after replating but did notdivide. Quiescent fibroblasts did not show staining. Keratinocytes,umbilical vein endothelial cells, and mammary epithelial cells allshowed increased staining with increased population doublings.Immortalized cells and terminally differentiated keratinocytes did notshow staining. Staining was performed on skin biopsies to test whethersenescence is observed in vivo. An age-dependent pattern in which anincreased number of cells showed staining with increased donor age wasobserved in the dermis and epidermis (Dinri et al., Proc. Nat. Acad.Sci. USA 92:9363-9367, 1995). The existence of an increase in the numberof senescent fibroblasts has been shown in the lungs of subjects withemphysema relative to subjects without emphysema (Müller et al., Resp.Res. 7:32-41, 2006).

Cellular senescence confers a number of functional changes on the cellthat likely have clinical relevance. Senescent endothelial cells secreteelevated levels of plasminogen activator inhibitor 1 (PAI-1; Kletsas etal., Ann. N. Y. Acad Sci. 908:11-25, 2000). Senescent fibroblasts overexpress collagenase and under express collagenase inhibitors (West etal., Exp Cell Res 184:138-147, 1989). Serial passages of humanfibroblasts from a 25 year old donor showed increased elastaseendopeptidase type activity (Homsy et al., Journal of InvestigativeDermatology 91:472-477, 1988). Endothelial cells obtained from tissueoverlying atherosclerotic plaques were observed to have a senescentmorphology and express increased levels of PAI-1 and intracellularadhesion molecule 1 and decreased levels of nitric oxide (Davis et al.,British Heart Journal 60:459-464, 1988; Arterloscler. Thromb.11:1678-1689, 1991; Finn et al., Circulation 105:1541-1544, 1976; Comiet al., Exp. Cell Res. 219:304-308, 1995; Chang et al., Proc. Nat. Acad.Sci. USA 92:11190-11194, 1995). Indirect evidence that cellularsenescence may play a role in cardiovascular disease also is provided bythe observation that shorter leukocyte telomere length is associatedwith an increased risk of premature myocardial infarction (Brouilette etal., Arteriosclerosis, Thrombosis, and Vascular Biology 23:842-846,2003).

Cancer cells are immortal, meaning that they can replicate indefinitelywithout exhibiting senescence. A preponderance of opinion in thescientific community says that the teleological purpose of senescence isto prevent cancer by limiting the number of cell divisions that canoccur. This view is supported by experiments in mice showing that p53knockout results in increased cancer incidence and severity. Indirectevidence that senescence suppresses cancer occurrence includes theobservations that oncogenes immortalize or extend cellular lifespan andtumor suppressors Rb and p53, which are critical for senescence, suffera loss of function mutation in cancer (Shay et al., Biochem. Biophys.Acta. 1071:1-7, 1991).

Senescent cells can also promote tumorigenesis. Senescent stromal cellsexpress tumor promoting as well as tumor suppressing factors that exerta paracrine effect on neighboring epithelial cells; such effects includemitogenicity and antiapoptosis (Chang et al., Proc. Nat. Acad. Sci. USA97(8):4291-4296, 2000). Senescent fibroblasts have been shown tostimulate premalignant and malignant epithelial cells but not normalepithelial cells to form tumors in mice; this occurred when as few as10% of the fibroblasts were senescent (Krtolica et al., Proc. Nat. Acad.Sci. USA 98:12072-12077, 2001). Tumor promoting factors secreted bysenescent cells are partly mediated by p21^(waf1/cip1/sdi1) (Roninson,Cancer Res. 63:2705-2715, 2003). A threshold of senescent stromal cellsappears to provide a milieu allowing adjacent premalignant epithelialcells to survive, migrate, and divide (Campisi, Nat. Rev. Cancer3:339-349, 2003).

In summary, cellular senescence does occur in vivo and is a likelysequel to environmental insults. Its prevalence increases with age atleast in some tissue compartments. Senescence confers functional changeson the cell which have been associated to some degree with variousage-related diseases (Chang et al., Proc. Nat. Acad. Sci. USA97(8):4291-4296, 2000). Senescent cells also contribute to tumorformation. There exists a need for agents that are capable of detectingsenescent cells in vivo and for treating or preventing diseases anddisorders related to or caused by cellular senescence.

SUMMARY OF THE INVENTION

This invention features compositions and methods for the detection andimaging of senescent cells in vitro or in vivo in order to predict therisk of diseases or disorders related to or caused by cellularsenescence (e.g., cancer occurrence, cancer metastasis, cardiovasculardisease, cerebrovascular disease, Alzheimer's disease, and emphysema).The invention also features compositions and methods for treating,preventing, or inhibiting the development or progression of diseases ordisorders related to or caused by cellular senescence (e.g., byadministering an agent that results in the death and removal of one ormore, all, or substantially all senescent cells or cells expressing oneor more senescence markers from an organism). The compositions andmethods can be administered in order to prevent, ameliorate, inhibit thedevelopment of, or treat diseases or disorders related to or caused bycellular senescence (e.g., cancer, cardiovascular disease,cerebrovascular disease, Alzheimer's disease, emphysema, osteoarthritis,and other age-related diseases). The invention features peptides whichbind with higher affinity to senescent cells than non-senescent cells.

In a first aspect, the invention features a peptide, polypeptide,antibody, or antibody fragment agent having an amino acid sequence setforth in any one of SEQ ID NOS:1-3 and 5-8 or a peptide, polypeptide,antibody, antibody fragment, or small molecule agent that is capable ofspecifically binding to an antigen having an amino acid sequence havingat least 20 amino acids with at least 80% sequence identity to any oneof the amino acid sequences set forth in SEQ ID NOS:11-23. In oneembodiment, the antigen has an amino acid sequence having at least 20amino acids of any one of the amino acid sequences set forth in SEQ IDNOS:11-23. In another embodiment, the antigen has any one of the aminoacid sequences set forth in SEQ ID NOS:11-23.

In embodiments of the first aspect of the invention, the agent includesa detectable label, a therapeutic agent, a chelating agent, or a linkermoiety. An agent of the first aspect of the invention can be indirectlyor directly attached to the detectable label. The linker moiety can havethe amino acid sequence GGGC, GGGS, or GG. A detectable label can be aradioactive agent, fluorescent agent, bioluminescent molecule, epitopetag, or heavy metal. Radioactive agents include iodine, astatine, andbromine labels that are attached to an amino acid of an agent of thefirst aspect of the invention. A radioactive agent can also betechnetium-99m. Fluorescent agents include fluorescein isothiocyanate(FITC), allophycocyanin (APC), phycoerythrin (PE), rhodamine,tetramethyl rhodamine isothiocyanate (TRITC), fluorescent protein (GFP),enhanced GFP (eGFP), yellow fluorescent protein (YFP), cyan fluorescentprotein (CFP), red fluorescent protein (RFP), and dsRed. Abioluminescent molecule can be luciferase. Epitope tags include c-myc,hemagglutinin, and histidine tags. A therapeutic agent can be acytotoxic agent, such as an alkylating agent, an antibiotic, anantineoplastic agent, an antimetabolic agent, a ribosomal activityinhibitor, an antiproliferative agent, a tubulin inhibitor, atopoisomerase I or II inhibitor, a growth factor, an hormonal agonist orantagonists, an apoptotic agent, an immunomodulator, a radioactiveagent, a phospholipase, or a cytotoxic peptide or lysin. A cytotoxicagent can also be ricin, doxorubicin, methotrexate, camptothecin,homocamptothecin, thiocolchicine, colchicine, combretastatin,combretastin A-4, podophyllotoxin, rhizoxin, rhizoxin-d, dolistatin,paclitaxel, CC 1065, ansamitocin p3, maytansinoid, streptolysin O,stoichactis toxin, phallolysin, staphylococcus alpha toxin, holothurinA, digitonin, melittin, lysolecithin, cardiotoxin, cerebratulus A toxin,or any derivative thereof. A chelating agent joined to an agent of thefirst aspect of the invention can be an ininocarboxylic reactive group,a polyaminopolycarboxylic reactive group, diethylenetriaminepentaaceticacid (DTPA), or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA). In any embodiment, the agent of the first aspect of theinvention can further include a pharmaceutically acceptable carrier. Anagent of the first aspect of the invention can specifically bind to asenescent cell, such as a senescent lung, breast, colon, prostate,gastric, hepatic, ovarian, esophageal, or bronchial epithelial orstromal cell, a senescent skin epithelial or stromal cell; a senescentglial cell; or a senescent vascular endothelial or stromal cell.

In a second aspect, the invention features a method of imaging asenescent cell-containing region in a mammal, such as a human, in vivo,by administering to the mammal an agent of the first aspect of theinvention that is joined to a detectable label, allowing the agent tobind senescent cells and allowing unbound agent to be cleared from thebody of said mammal, and obtaining an image of the senescentcell-containing region. Senescent cell-containing regions include thebreast, prostate, gastrointestinal tract, liver, lungs, intracranialspace, nasopharynx, oropharynx, larynx, esophagus, mediastinum, abdomenand pelvis, any region of the body containing peripheral vasculature,and the entire body. An image of the senescent cell-containing regioncan be obtained by scintigraphy.

In a third aspect, the invention features a method of predicting cancerrisk in a mammal, such as a human, by administering to the mammal anagent of the first aspect of the invention that is joined to adetectable label and predicting an elevated cancer risk in the mammal bydetecting binding of the agent to a senescent cell of the mammal. Themethod can be used to predict the risk of prostate cancer, colon cancer,lung cancer, squamous cell cancer of the head and neck, esophagealcancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer,ovarian cancer, and breast cancer.

In a fourth aspect, the invention features a method of treating orpreventing disease in a mammal, such as a human, by administering to themammal an agent of the first aspect of the invention joined to acytotoxic agent.

In a fifth aspect, the invention features a method of treating orpreventing disease in a mammal, such as a human, by administering to themammal a nucleic acid molecule encoding a cytotoxic agent and an agentof the first aspect of the invention. The nucleic acid molecule can beadministered in a vector, such as an adenoviral vector. In oneembodiment, the cytotoxic agent and agent of the first aspect of theinvention are expressed as a single polypeptide chain.

In either the fourth or fifth aspects of the invention, diseases thatcan be treated or prevented include cancer, age-related diseases,tobacco-related diseases, and skin wrinkles. Cancers that can be treatedor prevented include prostate cancer, colon cancer, lung cancer,squamous cell cancer of the head and neck, esophageal cancer,hepatocellular carcinoma, gastric cancer, pancreatic cancer, ovariancancer, and breast cancer. Age-related or tobacco-related diseasesinclude cardiovascular disease, cerebrovascular disease, peripheralvascular disease, Alzheimer's disease, osteoarthritis, cardiac diastolicdysfunction, benign prostatic hypertrophy, aortic aneurysm, andemphysema.

In a sixth aspect, the invention features a method for identifying apeptide or polypeptide capable of detecting senescent cells by (a)culturing cells to produce senescent cells, (b) exposing the senescentcells to a phage peptide library, (c) recovering the senescent cells andphage from said phage peptide library bound to said senescent cells, (d)eluting and amplifying bound phage to produce amplified phage, (e)repeating steps (a)-(d) one or more times, and (f) recovering a peptideor polypeptide expressed by the amplified phage that is capable ofdetecting senescent cells.

In a seventh aspect, the invention features a method for identifying anantibody or antibody fragment capable of specifically binding to asenescent cell-specific antigen by contacting an antibody or antibodyfragment with a polypeptide having at least 20 amino acids having atleast 80% amino acid sequence identity to any one of the amino acidsequences set forth in SEQ ID NOS:11-23, and identifying an antibody orantibody fragment that binds the polypeptide with a dissociationconstant of less than 10⁻⁷ M.

In an eighth aspect, the invention features a method for identifying anantibody or antibody fragment capable of specifically binding to asenescent cell-specific antigen by administering a polypeptide having atleast 20 amino acids having at least 80% amino acid sequence identity toany one of the amino acid sequences set forth in SEQ ID NOS:11-23 to amammal, allowing the mammal to generate a humoral immune response to thepolypeptide, and identifying an antibody or antibody fragment that bindsthe polypeptide with a dissociation constant of less than 10⁻⁷ M.Mammals suitable for the identification of antibodies that specificallybind senescent cell-specific antigens include mice, hamsters, rats,guinea pigs, chickens, goats, sheep, cows, horses, non-human primates,and humans.

In an ninth aspect, the invention features a method for identifying asmall molecule capable of specifically binding to a senescentcell-specific antigen by contacting a small molecule with a polypeptidehaving at least 20 amino acids having at least 80% amino acid sequenceidentity to any one of the amino acid sequences set forth in SEQ IDNOS:11-23 and identifying a small molecule that binds the polypeptidewith a dissociation constant of less than 10⁻⁷ M.

In an tenth aspect, the invention features a method of making anantibody or antibody fragment by recombinantly expressing a nucleic acidsequence that encodes an amino acid sequence having one or more of SEQID NOs:1-3 and 5-8, wherein the antibody or antibody fragmentspecifically binds a senescent cell.

In an eleventh aspect, the invention features a method of cellulartherapy performed by administering to a mammal, such as a human, in needthereof an agent of the invention prior to, concurrent with, orfollowing administration of a cellular therapeutic.

In an twelfth aspect, the invention features a method of cellulartherapy performed by contacting an agent of the invention to a donorcell, tissue, or organ prior to, concurrent with, or followingadministration of the cell, tissue, or organ to a mammal, such as ahuman. A donor cell, tissue, or organ can be an autologous, allogeneic,syngeneic, or xenogeneic cell, tissue, or organ. A donor cell can be astem cell, such as a hematopoietic, umbilical cord blood, totipotent,multipotent, or pluripotent stem cell.

In a thirteenth aspect, the invention features a kit containing an agentof the invention and one or more of a detectable label, a therapeuticagent, a chelating agent, or a linker moiety.

The term “about” is used herein to mean a value that is ±10% of therecited value.

By “administration” or “administering” is meant a method of providing adosage of an agent of the invention to a mammal (e.g., a human), wherethe route is, e.g., topical, oral, parenteral (e.g., intravenous,intraperitoneal, intrarterial, intradermal, intramuscular, orsubcutaneous injection, inhalation, optical drops, or implant), nasal,vaginal, rectal, or sublingual application in admixture with apharmaceutically acceptable carrier adapted for such use. The preferredmethod of administration can vary depending on various factors, e.g.,the components of the pharmaceutical composition, site of the potentialor actual disease (e.g., the location of lung, breast, colon, prostate,liver, brain, heart, etc.), and the severity of disease.

By “analog” is meant an agent that differs from, but is structurally,functionally, and/or chemically related to the reference agent. Theanalog may retain the essential properties, functions, or structures ofthe reference agent. Most preferably, the analog retains at least onebiological function of the reference agent. Generally, differences arelimited so that the structure or sequence of the reference agent and theanalog are similar overall. For example, a peptide analog and itsreference peptide may differ in amino acid sequence by one or more aminoacid substitutions, additions, and/or deletions, or the presence of oneor more non-naturally occurring amino acid residues, in any combination.An analog of a peptide or polypeptide of the invention may be naturallyoccurring, such as an allelic variant, or it may be a variant that isnot known to occur naturally. Non-naturally occurring analogs ofpeptides may be made by direct synthesis, by modification, or bymutagenesis techniques.

By “chelating agent” is meant a molecule that forms multiple chemicalbonds with a single metal atom. Prior to forming the bonds, thechelating agent has more than one pair of unshared electrons. The bondsare formed by sharing pairs of electrons with the metal atom. Chelatingagents include, for example, an iminodicarboxylic group or apolyaminopolycarboxylic group. Chelating agents may be attached to anagent of the invention using the methods generally described in Liu etal., Bioconjugate Chem. 12(4):653, 2001; Alter et al., U.S. Pat. No.5,753,627; and PCT Publication No. WO 91/01144; each of which is herebyincorporated by reference). An agent of the invention may be complexed,through its attached chelating agent, to a detectable label, therebyresulting in an agent that is indirectly labeled. Similarly, cytotoxicor therapeutic agents, may also be attached via a chelating group to anagent of the invention.

By “coupled” is meant the characteristic of a first molecule beingjoined to a second molecule by a covalent bond or through noncovalentintermolecular attraction.

By “cytotoxic agent” is meant any naturally occurring, modified, orsynthetic compound that is toxic to cells. Such agents are useful in thetreatment of neoplasms, and in the treatment of other symptoms ordiseases characterized by cell proliferation or a hyperactive cellpopulation. Cytotoxic agents can also be used to target undesirablecells or tissues other than neoplastic cells or tissues, e.g., senescentcells. Cytotoxic agents include, but are not limited to, alkylatingagents, antibiotics, antimetabolites, tubulin inhibitors, topoisomeraseI and II inhibitors, hormonal agonists or antagonists, immunomodulators,or agents that cause cell lysis including naturally occurring orsynthetic peptides. Cytotoxic agents may be cytotoxic when activated bylight or infrared (Photofrin, IR dyes; Nat. Biotechnol. 19(4):327-331,2001), may operate through other mechanistic pathways, or besupplementary potentiating agents.

By “detectable label” is meant any type of label which, when attached toan agent of the invention, renders the agent detectable. A detectablelabel may be toxic or non-toxic, and may have one or more of thefollowing attributes, without restriction: fluorescence (Kiefer et al.,WO 9740055), color, toxicity (e.g., radioactivity, e.g., a γ-emittingradionuclide, Auger-emitting radionuclide, β-emitting radionuclide, anα-emitting radionuclide, or a positron-emitting radionuclide),radiosensitivity, or photosensitivity. Although a detectable label maybe directly attached, for example, to an amino acid residue of an agentof the invention, or indirectly attached, for example, by beingcomplexed with a chelating group that is attached (e.g., linked via acovalent bond or indirectly linked) to an amino acid residue of an agentof the invention. A detectable label may also be indirectly attached toan agent of the invention by the ability of the label to be specificallybound by a second molecule. One example of this type of an indirectlyattached label is a biotin label that can be specifically bound by asecond molecule, streptavidin. The second molecule may also be linked toa moiety that allows neutron capture (e.g., a boron cage as describedin, for example, Kahl et al., Proc. Natl. Acad Sci. USA 87:7265-7269,1990).

A detectable label may also be a metal ion from heavy elements or rareearth ions, such as Gd³⁺, Fe³⁺, Mn³⁺, or Cr²⁺ (see, e.g., Curter,Invest. Radiol. 33(10):752-761, 1998). Preferred radioactive detectablelabels are radioactive iodine labels (e.g., ¹²²I, ¹²³I, ¹²⁴I, ¹²⁵I, or¹³¹I) that are capable of being coupled to each D- or L-Tyr or D- orL-4-amino-Phe residues present in the agents of the invention. Preferrednon-radioactive detectable labels are the many known dyes that arecapable of being coupled to NH₂-terminal amino acid residues.

Preferred examples of detectable labels that may be toxic to cellsinclude ricin, diphtheria toxin, and radioactive detectable labels(e.g., ¹²²I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ⁶⁴Cu, ⁶⁷Cu, ¹⁵³Sm, ¹⁶⁶Ho,¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, ²¹²Bi, ²²⁵Ac, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br,^(117m)Sn, ⁴⁷Sc, ¹⁰⁹Pd, ⁸⁹Sr, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴²Pr, ¹¹¹Ag, ¹⁶⁵Dy, ²¹³Bi,¹¹¹In, ^(114m)In ²⁰¹Ti, ^(195m)Pt, ¹⁹³Pt, ⁸⁶Y and ⁹⁰Y). These compounds,and others described herein may be directly or indirectly attached to anagent of the invention or its analogs. A toxic detectable label may alsobe a chemotherapeutic agent (e.g., camptothecins, homocamptothecins,5-fluorouracil or adriamycin), or may be a radiosensitizing agent (e.g.,paclitaxel, gemcitabine, fluoropyrimidine, metronitozil, or thedeoxycytidine analog 2′,2′ difluoro-2′-deoxycytidine (dFdCyd) to whichis directly or indirectly attached an agent or analog thereof of thepresent invention.

A detectable label, when coupled to an agent of the invention emits asignal that can be detected by a signal transducing machine. In somecases, the detectable label can emit a signal spontaneously, such aswhen the detectable label is a radionuclide. In other cases thedetectable label emits a signal as a result of being stimulated by anexternal field such as when the detectable label is a relaxivity metal.Examples of signals include, without limitation, gamma rays, X-rays,visible light, infrared energy, and radio waves. Examples of signaltransducing machines include, without limitation, gamma camerasincluding SPECT/CT devices, PET scanners, fluorimeters, and MagneticResonance Imaging (MRI) machines.

By “diagnostically effective amount” is meant a dose ofdetectably-labeled agent that, when administered internally to a mammal,is quantitatively sufficient to be detected by a signal transducingmachine external to the mammal (e.g., a gamma camera used in gammascintigraphy) but typically is quantitatively insufficient to produce apharmacological effect.

By “imaging agent” is meant a compound that, when administered to aliving subject, such as a mammal (e.g., a human), allows thevisualization of internal structures (e.g., cells, tissues, and organs)and, in some cases can provide information as to the function of a cell,tissue, or organ in the subject.

By “linker moiety” is meant a sequence of amino acid residues, e.g., atleast one, two, three, four, five, six, seven, eight, nine, ten,fifteen, twenty, thirty, forty, fifty, or more residues, that couples anagent of the invention (e.g., a peptide, polypeptide, protein, smallmolecule, antibody, or antibody fragment that target senescent cells)to, e.g., one or more of a detectable label, a therapeutic agent, and achelating agent.

By “senescent cell” is meant a cell that is metabolically active but ispermanently withdrawn from the cell cycle (see, e.g., Campisi, Cell120:513-522, 2005). Senescent cells do not replicate and possess one ormore of the following additional characteristics attributed to senescentcells: cell cycle arrest in the G1 phase; an enlarged, flattenedmorphology; increased granularity; staining for β-galactosidase activityat pH 6; senescence associated heterochromatic foci; and characteristicgene expression that is in part regulated by p16 and p21. Alternatively,a senescent cell is a cell that can be induced to become senescent(e.g., by stress) or that expresses cell-surface markers characteristicof senescent cells; these markers include senescent cell-specificantigens having the polypeptide sequences set forth in SEQ ID NOS:11-23.Senesecent cell-specific antigens are those peptides, polypeptides, orglycoproteins that are expressed on the cell surface of senescent cells,but are absent or only weakly expressed on the cell surface ofnon-senescent cells.

By “peptide” is meant a polymer that includes two or more amino acidsjoined to each other by a peptide bond or a modified peptide bond.“Peptide” refers to both short chain polymers, commonly referred to aspeptides, oligopeptides, or oligomers, having, e.g., about 10-50 linkedamino acid residues, and to longer polymers having up to about 100 aminoacid residues in length. Peptides may contain amino acids other than the20 gene-encoded amino acids, and linkages other than peptide bonds andmay include cyclic or branched peptides. “Peptides” include amino acidsequences modified either by natural processes, or by chemicalmodification techniques that are well known in the art. Modificationsmay occur anywhere in a polypeptide, including the peptide backbone, theamino acid side-chains, and the amino or carboxyl termini.

The notations used herein for the peptide amino acid residues are thoseabbreviations commonly used in the art. The less common abbreviationsAbu, Ava, β-Ala, hSer, Nle, Nva, Pal, Dab, and Dap stand for2-amino-butyric acid, amino valeric acid, beta-aminopropionic acid,homoserine, norleucine, norvaline, (2,3, or 4) 3-pyridyl-Ala,1,4-diaminobutyric acid, and 1,3-diaminopropionic acid, respectively. Inall aspects of the invention, it is noted that when amino acids are notdesignated as either D- or L-amino acids, the amino acid is either anL-amino acid or could be either a D- or L-amino acid. Examples ofpeptides of the invention include those peptides having the sequence of,or a sequence substantially identical to, the sequences set forth in SEQID NOs:1-3 and 5-8 and related sequences. These peptide sequences canalso be incorporated into a polypeptide, protein, antibody, or antibodyfragment.

By “reducing agent” is meant a chemical compound used to reduce anotherchemical compound by donating electrons, thereby becoming oxidized.

By “specifically binds” is meant that an agent of the inventionrecognizes and binds to a target (e.g., a senescent cell), but does notsubstantially recognize and bind to a non-target (e.g., non-senescentcells), both in vivo and in a sample, e.g., an in vitro biologicalsample, that includes, e.g., senescent cells. A desirable agent of theinvention specifically binds to senescent cells. Preferably, the agentsof the invention bind senescent cells with at least 2, 5, 10, 20, 100,or 1000 fold greater affinity than they bind to non-senescent cells.Alternatively, agents of the invention specifically bind to senescentcells with a dissociation constant less than 10⁶M, more preferably lessthan 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, 10⁻¹⁰M, 10⁻¹¹M, or 10⁻¹²M, and most preferablyless than 10⁻¹³M, 10⁻¹⁴M, or 10⁻¹⁵M.

By “substantial sequence identity” or “substantially identical” is meantthat a nucleic acid or amino acid sequence exhibits at least 50%,preferably 60%, 70%, 75%, or 80%, more preferably 85%, 90% or 95%, andmost preferably 99% identity to a reference amino acid sequence (e.g.,one or more of the sequences set forth in SEQ ID NOs:1-3 and 5-8). Foramino acid sequences, the length of comparison sequences will generallybe at least 5 amino acids, preferably at least 10 contiguous aminoacids, more preferably at least 15, 20, 25, 30, 40, 50, 60, 80, 90, 100,150, 200, 250, 300, or 350 contiguous amino acids, and most preferablythe full-length amino acid sequence. Sequence identity is typicallymeasured using BLAST® (Basic Local Alignment Search Tool) or BLAST®2with the default parameters specified therein (see, Altschul et al., J.Mol. Biol. 215:403-410, 1990); and Tatiana et al., FEMS Microbiol. Lett.174:247-250, 1999). This software program matches similar sequences byassigning degrees of homology to various substitutions, deletions, andother modifications. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine, valine,isoleucine, leucine; aspartic acid, glutamic acid, asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine.

By “therapeutic agent” is meant any compound that is used in thedetection, diagnosis or treatment of disease. Such compounds may benaturally-occurring, modified, or synthetic. A therapeutic agent may be,for example, an agent that causes apoptosis or necrosis of a cell (e.g.,a senescent cell) in an organism (e.g., a mammal, such as a human),thereby reducing the number of such cells in the organism. Therapeuticagents that reduce the number of senescent cells in an organism may be,e.g., alkylating agents, antibiotics, antimetabolites, hormonal agonistsor antagonists, anti- or pro-apoptotic agents, immunomodulators, orsupplementary potentiating agents.

By “treating, stabilizing, or preventing cancer” is meant causing areduction in the size of a tumor or in the number of cancer cells,slowing or preventing an increase in the size of a tumor or in cancercell proliferation, increasing the disease-free survival time betweenthe disappearance of a tumor or other cancer and its reappearance,preventing an initial or subsequent occurrence of a tumor or othercancer, or reducing an adverse symptom associated with a tumor or othercancer. In a desired embodiment, the percent of tumor or cancerous cellssurviving the treatment is at least 20, 40, 60, 80, or 100% lower thanthe initial number of tumor or cancerous cells, as measured using anystandard assay, such as those described herein. Desirably, the decreasein the number of tumor or cancerous cells induced by administration of acompound of the invention is at least 2, 5, 10, 20, or 50-fold greaterthan the decrease in the number of non-tumor or non-cancerous cells.Desirably, the methods of the present invention result in a decrease of20, 40, 60, 80, or 100% in the size of a tumor or number of cancerouscells as determined using standard methods. Desirably, at least 20, 40,60, 80, 90, or 95% of the treated subjects have a complete remission inwhich all evidence of the tumor or cancer disappears. Desirably, thetumor or cancer does not reappear after more than 5, 10, 15, or 20years.

By “treating, stabilizing, or preventing age-related diseases” is meantcausing a reduction in the number of symptoms, a decrease in severity ofany, all, or substantially all of the symptoms, a complete resolution ofany, all, or substantially all symptoms, or preventing the occurrence ofany, all, or substantially all symptoms associated with one or more ofthe age-related diseases including, but not limited to, cardiovasculardisease, cerebrovascular disease, peripheral vascular disease,Alzheimer's disease, osteoarthritis, cardiac diastolic dysfunction,benign prostatic hypertrophy, and cancers that increase in incidence andprevalence with increasing patient age such as cancer, e.g., breastcancer, prostate cancer, and colon cancer.

By “treating, stabilizing, or preventing tobacco-related diseases” ismeant causing a reduction in the number of symptoms, a decrease inseverity of any, all, or substantially all of the symptoms, a completeresolution of any, all, or substantially all symptoms, or preventing theoccurrence of any, all, or substantially all symptoms associated withone or more of the diseases associated with the use of smoking tobaccoas a risk factor, including, but are not limited to, cardiovasculardisease, cerebrovascular disease, peripheral vascular disease, aorticaneurysms, emphysema, esophageal cancer, lung cancer, and squamous cellcancers of the head and neck. Tobacco-related diseases also includediseases that are associated with the use of chewing tobacco, such assquamous cell cancers of the mouth.

Other features and advantages of the invention will be apparent from thefollowing description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the cytotoxicity of SenL (SEQ ID NO:4) whentested on senescent fibroblasts, non-senescent fibroblasts, andimmortalized prostate epithelial cells. No cell death was observed inimmortalized epithelium.

FIG. 2 is a graph showing the results of a WST assay used to measurecell proliferation following treatment with SenL on senescentfibroblasts, non-senescent fibroblasts, and immortalized prostateepithelial cells. The WST-1 assay depends on mitochondrial dehydrogenaselevels, and senescent cells have higher mitochondrial mass than theirnon-senescent counterparts (Martin-Ruiz et al., J. Biol. Chem.279(17):17826-33, 2004). Consequently, baseline values for senescentcells are higher than for the other cell types. Treatment with SenLshowed no significant effect on metabolic activity for any of the threecell types and therefore did not affect cell proliferation rates.

FIG. 3 is a graph showing the cytotoxicity of SenC (SEQ ID NO:5)conjugated to ricin A subunit as tested in senescent fibroblasts,non-senescent fibroblasts, and immortalized prostate epithelial cells.Significantly more senescent cells were killed than non-senescent cells,and no effect was observed on immortalized epithelium.

FIGS. 4A and 4B are fluorescent micrographs showing the specific bindingof a peptide agent of the invention. Senescent cell binding peptide SenC(SEQ ID NO:5) was conjugated to fluorescein and contacted with senescentfibroblasts (FIG. 4A) and non-senescent fibroblasts (FIG. 4B). Bothimages were acquired using 1/60 second exposure time. Senescent cellsshow perinuclear and cytoplasmic staining, indicating significantinternalization. Only faint surface staining is visible on thenon-senescent cells.

FIGS. 5A and 5B are immunofluorescent micrographs showing the cellsurface expression of cathepsin B on senescent cells (FIG. 5A) and lackof expression on non-senescent cells (FIG. 5B).

DETAILED DESCRIPTION OF THE INVENTION

The invention features agents of the invention (e.g., peptides,polypeptides, proteins, small molecules, antibodies, or antibodyfragments that target senescent cells) capable of specifically bindingto senescent cells. Thus, agents of the invention can include, e.g.,detectable labels, therapeutic agents, chelating agents, and cytotoxicagents, and can be used in the detection and treatment of diseases andconditions associated with cellular senescence.

In an embodiment, agents of the invention (e.g., peptides, polypeptides,proteins, small molecules, antibodies, or antibody fragments that targetsenescent cells) can be used to produce imaging agents by conjugation toa detectable label and used for medical imaging. Images obtained usingan imaging agent of the invention specifically show tissues, organs, andstructures in the body (e.g., a human body) that contain senescent cellsby means of, e.g., a signal emitted from the location of the cells,tissues, organs, and structures upon specific binding of the imagingagent to the senescent cells. The signal so obtained indicates thepresence of senescent cells and allows semi-quantitative estimation ofthe relative senescent cell content of tissues, organs, or structures.Structures, tissues, or organs that contain a threshold content ofsenescent cells are at an elevated risk for the subsequent developmentof diseases or disorders (e.g., cancer). Typically, a subject deemed atrisk for the subsequent development of diseases or disorders (e.g.,cancer) exhibit an increase of at least 0.5%, more preferably 1, 1.5, 2,2.5, 5, or 10%, and more preferably at least 15% or more in the level ofsenescent cells relative to a patient that does not have the disease ordisorder. Use of one or more of the imaging agents of the inventionallows for the determination of a patient's risk for developing adisease or disorder, such as cancer or a neurodegenerative disease.Patients determined to have an elevated risk of developing cancer orother diseases and disorders related to or caused by cellular senescenceby the use of the imaging agents of the invention can be aggressivelymonitored, and their cellular senescence related disease or disorder(e.g., a cancer) can thereby be detected earlier, facilitating earlytreatment. Surgical prophylactic treatment can also be administered ifnecessary.

One or more of the imaging agents of the invention can also be used todetermine the senescent cell content in the brain, which may include,e.g., senescent glial cells and senescent cerebrovascular cells, inorder to predict the patient's risk of developing, e.g., Alzheimer'sdisease or cerebrovascular disease. One or more of the imaging agents ofthe invention can also be used to predict the onset of vascular disease,including cardiovascular disease, by detecting the senescent cellcontent in one or more vascular structures in a patient. One or more ofthe imaging agents of the invention can also be used to predict the riskof developing emphysema by determining the senescent cell content in thelungs of a smoker.

One or more of the agents of the invention (e.g., peptides,polypeptides, proteins, small molecules, antibodies, or antibodyfragments that target senescent cells) can also be used to preparecompositions for therapeutic administration (e.g., to treat, stabilize,inhibit the development or progression of, or prevent age-relateddiseases, tobacco-related diseases, cancer, neurodegenerative diseases,and other diseases and disorders related to or caused by cellularsenescence) by coupling of the agents of the invention to, e.g.,therapeutic and/or cytotoxic agents. Senescent cells have been observedin skin to make up only a fraction of the stromal cell compartment evenin old donors (Dimri et al., Proc. Nat. Acad. Sci. USA 92:9363-9367,1995) and up to 16% of stromal fibroblasts in patients with emphysema(Bergner, Respiratory Res. 7:32, 2006), but senescent cells can exertclinically significant paracrine effects even when they compose only 10%of the content of stromal cells (Krtolica et al., Proc. Nat. Acad. Sci.USA 98:12072-12077, 2001). Thus, eliminating senescent cells whileleaving intact the majority of the cellular compartment from which thesenescent cells originate can ablate the harmful paracrine effects ofsenescent cells. The secretion of elevated levels of collagenase andelastase and depressed levels of collagenase inhibitors by senescentstromal cells implicates them in diseases or conditions that featurebreakdown or decrease in structural integrity of the extra cellularmatrix, including emphysema, aortic aneurysms, vascular disease,osteoarthritis, and skin wrinkling. Elimination of some, all, orsubstantially all senescent stromal cells in a patient (e.g., in atissue or organ of a patient) can attenuate the progression, decreasethe symptoms, or prevent the occurrence of these diseases andconditions. Senescent stromal cells have the ability to stimulatetumorigenesis and, by weakening the extracellular matrix, facilitatemetastasis. Thus, the elimination of some, all, or substantially all ofthe senescent stromal cells in a patient (e.g., in a tissue or organ ofa patient) can decrease the risk of tumorigenesis and metastasis (e.g.,by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more).

The senescent cell targeting agents of the invention (e.g., peptides,polypeptides, proteins, small molecules, antibodies, or antibodyfragments that target senescent cells) can be prepared by amino acidcoupling using solid phase peptide synthesis (SPPS). As is known in theart, the amino acids to be used as substrates to form the agents of theinvention are Fmoc-protected prior to incorporation into a peptide chain(see, e.g., Chanand White, FMOC Solid Phase Peptide Synthesis, APractical Approach, Oxford University Press, New York, 2003);incorporated herein by reference in its entirety). The standard couplingtechniques used to couple the amino acids in order to form the agents ofthe invention are known in the art (see, e.g., Chan and White, supra).For example, a polyamide-Rink resin can be prepared by loading apolyamide resin with Fmoc-Rink using chemical protocols well known inthe art (see, e.g., Chan and White, supra). The first amino acid in thepeptide sequence is coupled to the resin after removing Fmoc from theN-terminal amine of the resin using piperidine. Once the coupling iscomplete, the resin is washed and Fmoc is removed from the coupled aminoacid using piperidine. The resin is washed again, and the next aminoacid in the sequence is coupled to the previously coupled amino acid.This process is repeated using the necessary amino acids until thedesired peptide is formed. Following the coupling of the final aminoacid, the Fmoc group is removed using piperidine. The terminal amine canbe left as a free amine, or it can be acetylated. The peptide can becleaved from the resin using trifluoroacetic acid (TFA),triisopropylsilane, and water according to techniques known in the art(see, e.g., Chan and White, supra). The cleaved peptide is thenseparated from the residue by filtration. The TFA is typicallyevaporated to dryness followed by precipitation of the peptide withdiethyl ether. Typically, the final peptide product is purified usingHPLC. Mass spectrometry is used to verify that the desired peptide isobtained. The agents of the invention (e.g., peptides, polypeptides,proteins, small molecules, antibodies, or antibody fragments that targetsenescent cells) can be readily prepared by automated solid phasepeptide synthesis using any one of a number of well-known, commerciallyavailable automated synthesizers, such as the Applied Biosystems ABI433A peptide synthesizer.

The agents of the invention (e.g., peptides, polypeptides, proteins,small molecules, antibodies, or antibody fragments that target senescentcells) can be labeled for fluorescence detection by labeling the agentwith a fluorophore, such as rhodamine or fluorescein, using techniqueswell known in the art (see, e.g., Lohse et al., Bioconj. Chem.8:503-509, 1997). The senescent cell targeting agents of the invention(e.g., peptides, polypeptides, proteins, small molecules, antibodies, orantibody fragments that target senescent cells) can also be labeled witha radioactive metal or a relaxivity metal by coupling the agent to ametal chelating agent that chelates a radioactive metal or relaxivitymetal. Examples of chelating agents include, but are not limited to,ininocarboxylic and polyaminopolycarboxylic reactive groups,diethylenetriaminepentaacetic acid (DTPA), and1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Thechelating agent can be coupled via its amino acid side chain directly tothe agents of the invention. Alternatively, an intervening amino acidsequence can be coupled using SPPS to both the agents of the inventionand the chelating agent.

Senescent Cell-Specific Antigens

The inventors have discovered that senescent cells express senescentcell-specific antigens. These antigens can be specifically targeted andbound by the diagnostic or therapeutic (e.g., cytotoxic) agents of theinvention, as discussed herein. The specific binding of the diagnosticor therapeutic agents of the invention only to senescent cells and notnon-senescent cells reduces the incidence of inaccurate diagnoses (e.g.,using diagnostic agents) or bystander cell injury (e.g., resulting fromcytotoxic agents).

Senescent cell-specific antigens that can be targeted by agents of theinvention include, e.g., mutant beta-actin (SEQ ID NO:11; GI: 28336) andbeta-actin (ACTB) protein (SEQ ID NO:12; GI: 15277503), which wereidentified as cell-surface, senescent-specific antigens occurring inboth replicatively senescent and stress-induced prematurely senescentcells, drug resistance-related protein LRP (SEQ ID NO:13; GI: 1097308)and major vault protein (MVP; SEQ ID NO:14; GI: 19913410), which wereidentified as senescence-specific surface proteins in the replicativelysenescent cells only, and thyroid hormone binding protein precursor (SEQID NO:15; GI: 339647), unnamed protein product (SEQ ID NO:20; GI:35655), prolyl 4-hydroxylase, beta subunit precursor (P4HB; SEQ IDNO:16; GI: 20070125), chain A, human protein disulfide isomerase (PDI;SEQ ID NO:17; GI: 159162689), electron-transfer-flavoprotein, betapolypeptide (ETFB; SEQ ID NO:18; GI: 4503609), unnamed protein product(SEQ ID NO:21; GI: 158257194), unnamed protein product (SEQ ID NO:22;GI: 158259937), ATP synthase, H⁺ transporting, mitochondrial F1 complex,alpha subunit precursor (SEQ ID NO:19; GI: 4757810), and cathepsin B(CTSB; SEQ ID NO:23), which were identified on the cell surface ofstress induced prematurely senescent cells.

Antibodies that specifically bind to the senescent cell-specificantigens described herein are listed in Table 1. These antibodies, andany other polypeptide, antibody, antibody fragment, or small moleculethat specifically binds a senescent cell-specific antigen or fragmentthereof, can be incorporated in a diagnostic or therapeutic agent of theinvention, as discussed herein. In addition, the antibodies listed inTable 1 can be modified, e.g., by humanization, according to knownmethods, as is discussed below, for use in the diagnosis and treatmentof disease in a mammal, e.g., a human. Alternatively, antibodies againstthe senescent cell-specific antigens described herein for use in thediagnosis and treatment of disease in a mammal, e.g., a human, can beproduced according to methods known in the art, as is discussed below.

TABLE I Senescent Cell-Specific Antigen SEQ ID NO: Anti-SenescentCell-Specific Antigen Antibodies Mutant beta-actin 11 See, e.g., Leavittet al., Mol Cell Biol. 7: 2467-2476 (1987); Lin et al., PNAS USA 82:6995-6999 (1985). Beta-actin (ACTB) protein 12 Clone AC-15 (mouseanti-human beta-actin antibody); Clone mAbcam 8226 (mouse anti-humanbeta-actin antibody). Drug resistance-related 13 Clone 1032 (mouseanti-human LRP antibody); rabbit anti-human protein LRP LRP antibody(see, e.g., Kitazono et al., J. Natl. Cancer Inst. 91: 1647-1653(1999)). Major vault protein (MVP) 14 Clone 1014 (mouse anti-human majorvault protein antibody); clone 2Q431(mouse anti-human major vaultprotein antibody). Thyroid hormone binding 15 See, e.g., Cheng et al.,J. Biol. Chem. 262: 11221-11227 (1987). protein precursor Prolyl4-hydroxylase, beta 16 Clone P4HB (Abcam Cat. No. ab70415; mouseanti-human prolyl subunit precursor (P4HB) 4-hydroxylase, beta subunitprecursor antibody). Chain A, human protein 17 Abcam Cat. No. ab48167(rabbit anti-human PDI antibody); disulfide isomerase (PDI) clone 1D3(Santa Cruz Cat. No. sc-59640; mouse anti-human PDI antibody).Electron-transfer- 18 Abcam Cat. No. ab73986 (rabbit anti-human ETFPantibody. flavoprotein, beta polypeptide (ETFP) ATP synthase, H⁺ 19Clone 15H4 (Abcam Cat. No. ab14748; mouse anti-human transporting,mitochondrial ATP5A); see also, e.g., Vantourout et al., Mol. Immunol.45: 485-492 F1 complex, alpha subunit (2008). precursor Unnamed proteinproduct 20 See, e.g., Pihlajaniemi et al., EMBO J. 6: 643-649 (1987).(GI: 35655) Unnamed protein product 21 See, e.g., Altschul et al.,Nucleic Acids Res. 25: 3389-3402 (GI: 158257194) (1997) Unnamed proteinproduct 22 See e.g., Heidebrecht et al., Mol Cancer Res. 1: 271-9 (2003)(GI: 158259937) Cathepsin B (CTSB) 23 Clone ZZ12 (mouse anti-humancathepsin B antibody); clone S- 12 (goat anti-human cathepsin Bantibody).

Small Molecules of the Invention

The invention also features small molecules that can be useddiagnostically or therapeutically based on their ability to specificallybind senescent cells. For example, small molecules of the inventioninclude those capable of specifically binding to one or more of thesenescent cell-specific antigens described herein and those capable ofmimicking the physical, chemical, biological, and/or targetingcharacteristics of SEQ ID NOs:1-3 and 5-8 and peptides that aresubstantially identical. Small molecules of the invention can be labeledor fused to diagnostic or therapeutic linkers, markers, cytotoxicagents, or other agents of the invention to aid in the diagnosis ortreatment of diseases or disorders related to or caused by cellularsenescence. Furthermore, small molecules of the invention canspecifically bind a polypeptide that has at least 80% (80%, 90%, 95%,99%, or 100%) amino acid sequence identity to any one of the amino acidsequences set forth in SEQ ID NOS:11-23, or a fragment thereof.

Methods of Screening Small Molecules for Binding to Senescent Cells

The invention also features methods for the high throughput screening(HTS) of candidate small molecule agents of the invention for theirability to bind senescent cells or senescent cell-specific antigens,including, but not limited to, the polypeptides set forth in SEQ IDNOS:11-23, or fragments thereof. Candidate small molecules will also bescreened for their ability to specifically bind senescent cells. Ingeneral, candidate small molecules preferably bind target sequences witha dissociation constant less than 10⁻⁶ M for further consideration as anagent of the invention.

Senescent cells of any origin can be used in HTS binding assays andmethods. In general, fluorescence and luminescence based assays (e.g.,ELISA, colorimetric assays) are used to measure binding affinities ofcandidate small molecules contacted against single or multiple targetsenescent cells. Upon the identification of a candidate small moleculefrom a first screening process, further scrutiny of the binding affinityand ability of the candidate small molecule can be by means of a second,different HTS assay. This could be accomplished, for example, bycontacting the candidate small molecule with alternate senescent cellpopulations to more precisely determine the binding affinity of themolecule. A discussion of HTS methodologies is found in, e.g., Verkman,“Drug discovery in academia,” Am. J. Physiol. Cell Physiol. 286,C465-C474 (2004) and Dove, “Screening for content—the evolution of highthroughput,” Nat. Biotechnol. 21:859-864 (2003). Examples of HTSscreening methods for the discovery of useful small molecule agents arefound in, e.g., U.S. Pat. Nos. 7,279,286 and 7,276,346, which areincorporated by reference herein.

Candidate small molecules that have undergone HTS screening may befurther modified to empirically improve senescent cell bindingaffinities according to the design considerations discussed below.

Small Molecule Design

Small molecules of the invention can also be generated according to theprinciples of rational design. Computer modeling technology allowsvisualization of the three-dimensional atomic structure of a selectedmolecule and the design of new compounds that will interact withsenescent cells or senescent cell-specific antigens (e.g., the senescentcell-specific antigens discussed above (set forth in SEQ ID NOS:11-23),or fragments thereof). The three-dimensional construct typically dependson data from x-ray crystallographic analyses or NMR imaging of theselected molecule or epitope. A computer graphics system enablesprediction of how a candidate small molecule compound will bind to thetarget senescent cell and allows experimental manipulation of thestructures of the small molecule and target protein to perfect bindingspecificity. A prediction of what the molecule-protein interaction willbe when small changes are made in one or both can be determined by usingmolecular mechanics software and computationally intensive computers. Anexample of a molecular modeling system described generally aboveincludes the CHARMm and QUANTA programs (Polygen Corporation, Waltham,Mass.). CHARMm performs the energy minimization and molecular dynamicsfunctions, while QUANTA performs the construction, graphic modeling andanalysis of molecular structure. QUANTA allows interactive construction,modification, visualization, and analysis of the behavior of moleculesthat intact with each other. Another molecular modeling program that canbe used to identify small molecules for use in the methods of theinvention is DOCK (Kuntz Laboratory, UCSF).

Small Molecule Synthesis

Small molecules of the invention can be organic or inorganic compounds,and even nucleic acids. Specific binding to senescent cells can beachieved by including chemical groups having the correct spatiallocation and charge in the small molecule. In a preferred embodiment,compounds are designed with hydrogen bond donor and acceptor sitesarranged to be complementary to the targeted molecule or epitope. Anagent is formed with chemical side groups ordered to yield the correctspatial arrangement of hydrogen bond acceptors and donors when the agentis in a specific conformation induced and stabilized by binding to thetarget molecule or epitope. Additional binding forces such as ionicbonds and Van der Waals interactions can also be considered whensynthesizing a small molecule of the invention. The likelihood offorming the desired conformation can be refined and/or optimized usingmolecular computational programs.

Organic compounds can be designed to be rigid, or to present hydrogenbonding groups on edge or plane, which can interact with complementarysites. Rebek, Science 235, 1478-1484 (1987) and Rebek et al., J. Am.Chem. Soc. 109, 2426-2431 (1987) have summarized these approaches andthe mechanisms involved in binding of compounds to regions of proteins.

Synthetic methods can be used by one skilled in the art to make smallmolecules that interact with functional groups of proteins,glycoproteins, or epitopes present on the exterior or in the interior ofsenescent cells.

Preparation of Antibody Compositions of the Invention

The invention also provides antibody compositions that include one ormore of the sequences set forth in SEQ ID NOS:1-3 and 5-8, and peptidessubstantially identical thereto, which can be included in, e.g., one ormore of the complementarity determining regions (CDRs) of the antibodycompositions. The invention further provides antibody compositions thatspecifically bind a senescent cell-specific antigen having at least 80%(e.g., 80%, 90%, 95%, 99%, or 100%) amino acid sequence identity to anyone of the senescent cell-specific antigens set forth in SEQ IDNOS:11-23, or a fragment thereof.

The antibodies of the invention may be recombinant (e.g., chimeric orhumanized), synthetic, or naturally occurring. Antibodies or antibodyfragments of the invention can be recombinantly produced, or identifiedand isolated from a mammal that has been induced to produce or thatnaturally-produces antibodies that specifically bind to senescentcell-specific antigens. For example, a polypeptide having at least 80%(e.g, 85%, 90%, 95%, 99%, or 100%) amino acid sequence identity to oneof the senescent cell-specific antigens set forth in SEQ ID NOS:11-23can be administered (e.g., by injection) to a mammal (e.g., a mouse,rat, hamster, guinea pig, sheep, goat, cow, horse, non-human primate, orhuman) to provoke a humoral immune response against the immunizingsenescent cell-specific antigen. Blood, plasma, or ascites from theimmunized mammal can be screened to identify or harvest antibodies thatspecifically bind the immunizing senenescent cell-specific antigen. Ifdesired, the amino acid and/or nucleic acid sequences of the antibodiesproduced can be determined. Furthermore, all or a portion of the nucleicacid sequences of the antibodies can be incorporated into a vector(e.g., an expression vector or a viral vector) for expression of theantibody in a cell of a subject (e.g., a mammal, such as a human)following administration of the vector to the subject, or any other usesconsistent with the methods described herein.

The invention features complete antibodies, diabodies, bi-specificantibodies, antibody fragments, Fab fragments, F(ab′)2 molecules, singlechain Fv (scFv) molecules, tandem scFv molecules, or antibody fusionproteins. Antibodies of the invention include, e.g., the IgG, IgA, IgM,IgD, and IgE isotypes. Antibodies of the invention contain one or moreCDRs or binding peptides that bind to proteins, glycoproteins, orepitopes present on the exterior or in the interior of senescent cells.

Many of the antibodies, or fragments thereof, described herein canundergo non-critical amino-acid substitutions, additions or deletions inboth the variable and constant regions without loss of bindingspecificity or effector functions, or intolerable reduction of bindingaffinity (i.e., below about 10⁻⁷ M). Usually, an antibody incorporatingsuch alterations exhibits substantial sequence identity to a referenceantibody from which it is derived. Occasionally, a mutated antibody canbe selected having the same specificity and increased affinity comparedwith a reference antibody from which it was derived. Phage-displaytechnology offers powerful techniques for selecting such antibodies.See, e.g., Dower et al. (WO 91/17271), McCafferty et al. (WO 92/01047),and Huse (WO 92/06204), each of which is incorporated by referenceherein.

Antibody Fragments

In another embodiment of the invention, an agent of the invention is afragment of an intact antibody described herein. Antibody fragmentsinclude separate variable heavy chains, variable light chains, Fab,Fab′, F(ab′)₂, Fabc, and Fv. Fragments can be produced by enzymatic orchemical separation of intact immunoglobulins. For example, a F(ab′)₂fragment can be obtained from an IgG molecule by proteolytic digestionwith pepsin at pH 3.0-3.5 using standard methods such as those describedin Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPubs., N.Y. (1988). Fab fragments may be obtained from F(ab′)₂ fragmentsby limited reduction, or from whole antibody by digestion with papain inthe presence of reducing agents. Fragments can also be produced byrecombinant DNA techniques. Segments of nucleic acids encoding selectedfragments are produced by digestion of full-length coding sequences withrestriction enzymes, or by de novo synthesis. Often fragments areexpressed in the form of phage-coat fusion proteins. This manner ofexpression is advantageous for affinity-sharpening of antibodies.

Humanized Antibodies

The invention also includes humanized antibodies in which one or more ofthe CDRs are derived from a non-human antibody sequence (e.g., thoseantibodies described in Table 1), and one or more, but preferably all,of the CDRs bind specifically to a protein, glycoprotein, or epitopepresent on the exterior or in the interior of a senescent cell (e.g.,the senescent cell-specific antigens described above (SEQ ID NOS:11-23),or a fragment thereof).

A humanized antibody contains constant framework regions derivedsubstantially from a human antibody (termed an acceptor antibody), aswell as, in some instances, a majority of the variable region derivedfrom a human antibody. One or more of the CDRs (all or a portionthereof, as well as discreet amino acids surrounding one or more of theCDRs) are provided from a non-human antibody, such as a mouse antibody.The constant region(s) of the antibody, may or may not be present.Humanized antibodies provide several advantages over non-humanizedantibodies for therapeutic or diagnostic use in humans. These include:

1) The human immune system should not recognize the framework orconstant region of the humanized antibody as foreign, and therefore theantibody response against such an injected antibody should be less thanagainst a totally foreign mouse antibody or a partially foreign chimericantibody;

2) Because the effector portion of the humanized antibody is human, itmay interact better with other parts of the human immune system; and

3) Injected mouse antibodies have been reported to have a much shorterhalf-life in the human circulation than the half-life exhibited bynormal human antibodies (see, e.g., Shaw et al., J. Immunol.138:4534-4538 (1987)). Injected humanized antibodies have a half-lifeessentially equivalent to naturally occurring human antibodies, allowingsmaller and less frequent doses.

The substitution of one or more, e.g., mouse CDRs into a human variabledomain framework is most likely to result in retention of their correctspatial orientation if the human variable domain framework adopts thesame or similar conformation to the mouse variable framework from whichthe CDRs originated. This is achieved by obtaining the human variabledomains from human antibodies whose framework sequences exhibit a highdegree of sequence identity with the murine variable framework domainsfrom which the CDRs were derived. The heavy and light chain variableframework regions can be derived from the same or different humanantibody sequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See, e.g., Kettleborough et al., ProteinEngineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971(1993).

Suitable human antibody sequences are identified by computer comparisonsof the amino acid sequences of the mouse variable regions with thesequences of known human antibodies. The comparison is performedseparately for heavy and light chains but the principles are similar foreach.

Methods of preparing chimeric and humanized antibodies and antibodyfragments are described in U.S. Pat. Nos. 4,816,567, 5,530,101,5,622,701, 5,800,815, 5,874,540, 5,914,110, 5,928,904, 6,210,670,6,677,436, and 7,067,313 and U.S. Patent Application Nos. 2002/0031508,2004/0265311, and 2005/0226876. Preparation of antibody or fragmentsthereof is further described in U.S. Pat. Nos. 6,331,415, 6,818,216, and7,067,313. Each of these patents is incorporated herein by reference.

Diagnostic Agents Coupled to Agents of the Invention

Agents of the invention (e.g., peptides, polypeptides, proteins, smallmolecules, antibodies, or antibody fragments that target senescentcells) can be joined or coupled to any molecule that can be used tolabel, detect, identify, screen, or diagnose senescent cells in a mammal(e.g., a human). Diagnostic and therapeutic (e.g., cytotoxic) agents ofthe invention that include a detectable label can be administered in apharmaceutical compound to a subject (e.g., a cancer patient) or thesecompositions can be encoded by a vector (e.g., a viral vector) andexpressed in a cell of the subject following administration.

An agent of the invention that incorporates a detectable label can beused, for example, to type, grade, and track the metastasis of a tumorin a subject having cancer. The diagnostic agent can also include atherapeutic agent (e.g., cytotoxic) of the invention. In this case, thediagnostic agent facilitates the detection or measurement, or binding tosenescent cells, of the therapeutic agent after administration to asubject.

A diagnostic or therapeutic agent of the invention that includes adetectable label can be produced recombinantly in vitro. In oneembodiment of the invention, the detectable label is capable ofdetection based on an intrinsic property, e.g., fluorescence andbioluminescence, or based on its ability to bind with another moleculecapable of being detected (e.g., an epitope tag, such as c-myc,hemagglutinin, and histamine tags). Peptides, polypeptides, or proteinswith diagnostic properties can be altered (e.g., by making amino acidsubstitutions, mutations, truncations, or additions) to facilitateincorporation into an agent of the invention. Desirable alterationsinclude, for example, changes to the amino acid sequence that facilitateprotein expression, longevity, cell secretion, and detectability.

The invention also features a nucleic acid molecule encoding a peptidicdetectable label as a fusion protein with an agent of the invention; thenucleic acid molecule can be incorporated into a vector (e.g., anexpression vector), such that, upon expression of the agent of theinvention in a cell transfected or transduced with the vector,expression of the detectable label and agent of the invention areoperably linked (e.g., fused, contiguously-joined, or tetheredtogether).

Molecules that can be used as the detectable label as discussed hereininclude, but are not limited to, radioactive agents, fluorescent agents,bioluminescent molecules, epitope tags, and heavy metals, each of whichis discussed in detail below.

Fluorescent agents include fluorochromes, such as fluoresceinisothiocyanate (FITC), allophycocyanin (APC), phycoerythrin (PE),rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC). Otherfluorescent molecules include green fluorescent protein (GFP; SEQ IDNO:27), enhanced GFP (eGFP), yellow fluorescent protein (SEQ ID NO:28;YFP), cyan fluorescent protein (SEQ ID NO:29; CFP), and red fluorescentprotein (SEQ ID NO:30; RFP or DsRed). Each of these fluorescentmolecules can be used as detectable label in a diagnostic agent of theinvention. Peptidic fluorescent molecules can be recombinantly expressedin a cell (e.g., a blood cell, such as a lymphocyte) followingtransfection or transduction of the cell with an expression vector thatencodes the nucleotide sequence of the fluorescent molecule. Uponexposure of the fluorescent molecule to a stimulating frequency oflight, the fluorescent molecule will emit light at a low, medium, orhigh intensity that can be observed by eye under a microscope or by anoptical imaging device. Exemplary fluorescent molecules suitable for useas the detectable label in a diagnostic agent of the invention aredescribed in, e.g., U.S. Pat. Nos. 7,417,131 and 7,413,874.

Bioluminescent molecules can also be used as a detectable labelincorporated into a diagnostic agent of the invention. Bioluminescentmolecules, such as luciferase (e.g., firefly (SEQ ID NO:31), Renilla(SEQ ID NO:32), and Omphalotus luciferase) and aequorin, emit light aspart of a chemical reaction with a substrate (e.g., luciferin andcoelenterazine). In one embodiment of the invention, a vector encoding aluciferase gene provides for the in vivo, in vitro, or ex vivo detectionof cells (e.g., blood cells, such as lymphocytes) that have beentransduced or transfected with an agent of the invention. Exemplarybioluminescent molecules suitable for use as the detectable label in adiagnostic agent of the invention, and methods for their use aredescribed in, e.g., U.S. Pat. Nos. 5,292,658, 5,670,356, 6,171,809, and7,183,092.

Epitope tags are short amino acid sequences, e.g., 5-20 amino acidresidues in length, that can be incorporated into an agent of theinvention as a detectable label to allow for detection once expressed ina cell, secreted from the cell, or bound to a target cell (e.g., asenescent cell). An agent of the invention that incorporates an epitopetag as a diagnostic agent can be detected by virtue of its interactionwith an antibody, antibody fragment, or other binding molecule specificfor the epitope tag. Nucleotide sequences encoding the epitope tag areproduced either by cloning appropriate portions of natural genes or bysynthesizing a polynucleotide that encodes the epitope tag. An antibody,antibody fragment, or other binding molecule that binds an epitope tagcan directly incorporate its own detectable label (e.g., a fluorochrome,radiolabel, heavy metal, or enzyme such as horseradish peroxidase) orserve as a target for a secondary antibody, antibody fragment, or otherbinding molecule that incorporates such a label. Exemplary epitope tagsthat can be used as a detectable label include c-myc (SEQ ID NO:24),hemagglutinin (HA; SEQ ID NO:25), and histidine tag (His₆; SEQ IDNO:26). Furthermore, fluorescent (e.g., GFP) and bioluminescentmolecules, discussed above, can also serve as epitope tags, asantibodies, antibody fragments, and other binding molecules arecommercially available for the detection of these moieties.

Agents of the invention (e.g., peptides, polypeptides, proteins, smallmolecules, antibodies, or antibody fragments that target senescentcells) can also be coupled to a chelating agent to form diagnosticagents of the invention. Diagnostic agents can be prepared by variousmethods depending upon the chelator chosen. A portion of an agent of theinvention (e.g., a portion of a peptide, polypeptide, protein, smallmolecule, antibody, or antibody fragment) can be isolated from a naturalsource, recombinantly produced, or synthesized. The portion of an agentof the invention is most conveniently prepared by techniques generallyestablished in the art of peptide synthesis, such as the solid-phaseapproach. Solid-phase synthesis involves the stepwise addition of aminoacid residues to a growing peptide chain that is linked to an insolublesupport or matrix, such as polystyrene. The C-terminus residue of thepeptide is first anchored to a commercially available support with itsamino group protected with an N-protecting agent such as at-butyloxycarbonyl group (tBoc) or a fluorenylmethoxycarbonyl (FMOC)group. The amino-protecting group is removed with suitable deprotectingagents such as TFA in the case of tBOC or piperidine for FMOC and thenext amino acid residue (in N-protected form) is added with a couplingagent such as dicyclocarbodiimide (DCC). Upon formation of a peptidebond, the reagents are washed from the support. After addition of thefinal residue, the agent is cleaved from the support with a suitablereagent, such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF).

Agents of the invention (e.g., peptides, polypeptides, proteins, smallmolecules, antibodies, or antibody fragments that target senescentcells) and chelator components can be coupled to form a conjugate byreacting the free amino group of the threonine residue of a peptideportion of an agent of the invention with an appropriate functionalgroup of the chelator, such as a carboxyl group or activated ester. Forexample, a conjugate may incorporate the chelatorethylenediaminetetraacetic acid (EDTA), common in the art ofcoordination chemistry, when functionalized with a carboxyl substituenton the ethylene chain. Synthesis of EDTA derivatives of this type arereported in Arya el al., Bioconjugate Chemistry, 2:323, 1991), whereinthe four coordinating carboxyl groups are each blocked with a t-butylgroup while the carboxyl substituent on the ethylene chain is free toreact with the amino group of a peptide portion of the agent of theinvention, thereby forming a conjugate.

A conjugate may incorporate a metal chelator component that is peptidic,i.e., compatible with solid-phase peptide synthesis. In this case, thechelator may be coupled to the agent of the invention (e.g., peptides,polypeptides, proteins, small molecules, antibodies, or antibodyfragments that target senescent cells) in the same manner as EDTAdescribed above or more conveniently the chelator and agent of theinvention are synthesized in toto starting from the C-terminal residueof the agent and ending with the N-terminal residue of the chelator.

Conjugates may further incorporate a linking group component that servesto couple the agent of the invention (e.g., peptides, polypeptides,proteins, small molecules, antibodies, or antibody fragments that targetsenescent cells) to the chelator while not adversely affecting eitherthe targeting function of the agent or the metal binding function of thechelator. Suitable linking groups include amino acid chains and alkylchains functionalized with reactive groups for coupling to both theagent and the chelator. An amino acid chain is the preferred linkinggroup when the chelator is peptidic so that the conjugate can besynthesized in toto by solid-phase techniques.

An alkyl chain linking group may be incorporated in the conjugate byreacting the amino group of the threonine residue of a peptide portionof an agent of the invention (e.g., peptides, polypeptides, proteins,small molecules, antibodies, or antibody fragments that target senescentcells) with a first functional group on the alkyl chain, such as acarboxyl group or an activated ester. Subsequently the chelator isattached to the alkyl chain to complete the formation of the conjugateby reacting a second functional group on the alkyl chain with anappropriate group on the chelator. The second functional group on thealkyl chain is selected from substituents that are reactive with afunctional group on the chelator while not being reactive with thethreonine residue of the agent. For example, when the chelatorincorporates a functional group such as a carboxyl group or an activatedester, the second functional group of the alkyl chain linking group canbe an amino group. It will be appreciated that formation of theconjugate may require protection and deprotection of the functionalgroups present in order to avoid formation of undesired products.Protection and deprotection are accomplished using protecting groups,reagents, and protocols common in the art of organic synthesis.Particularly, protection and deprotection techniques employed in solidphase peptide synthesis described above may be used.

An alternative chemical linking group to an alkyl chain is polyethyleneglycol (PEG), which is functionalized in the same manner as the alkylchain described above for incorporation in the conjugates. It will beappreciated that linking groups may alternatively be coupled first tothe chelator and then to the agent of the invention (e.g., peptides,polypeptides, proteins, small molecules, antibodies, or antibodyfragments that target senescent cells).

In accordance with one aspect of the invention, agent-chelatorconjugates of the invention incorporate a diagnostically useful metalcapable of forming a complex. Suitable metals include, e.g.,radionuclides, such as technetium and rhenium in their various forms(e.g., ^(99m)TcO³⁺, ^(99m)TcO₂ ⁺, ReO³⁺, and ReO₂ ⁺). Incorporation ofthe metal within the conjugate can be achieved by various methods commonin the art of coordination chemistry. When the metal is technetium-99 m,the following general procedure may be used to form a technetiumcomplex. An agent-chelator conjugate solution is formed initially bydissolving the conjugate in aqueous alcohol such as ethanol. Thesolution is then degassed to remove oxygen then thiol-protecting groupsare removed with a suitable reagent, for example, with sodium hydroxide,and then neutralized with an organic acid, such as acetic acid (pH6.0-6.5). In the labeling step, a stoichiometric excess of sodiumpertechnetate, obtained from a molybdenum generator, is added to asolution of the conjugate with an amount of a reducing agent such asstannous chloride sufficient to reduce technetium and heated. Thelabeled conjugate may be separated from contaminants ^(99m)TcO₄ ⁻ andcolloidal ^(99m)TcO₂ chromatographically, for example, with a C-18 SepPak cartridge.

In an alternative method, labeling of the agent of the invention can beaccomplished by a transchelation reaction. The technetium source is asolution of technetium complexed with labile ligands facilitating ligandexchange with the selected chelator. Suitable ligands for transchelationinclude tartarate, citrate, and heptagluconate. In this instance thepreferred reducing reagent is sodium dithionite. It will be appreciatedthat the conjugate may be labeled using the techniques described above,or alternatively the chelator itself may be labeled and subsequentlycoupled to the agent of the invention to form the conjugate; a processreferred to as the “prelabeled ligand” method.

Another approach for labeling agents of the invention (e.g., peptides,polypeptides, proteins, small molecules, antibodies, or antibodyfragments that target senescent cells) involves immobilizing theagent-chelator conjugate on a solid-phase support through a linkage thatis cleaved upon metal chelation. This is achieved when the chelator iscoupled to a functional group of the support by one of the complexingatoms. Preferably, a complexing sulfur atom is coupled to the supportwhich is functionalized with a sulfur protecting group such asmaleimide.

When labeled with a diagnostically useful metal, agent-chelatorconjugates of the invention can be used to detect tissue at risk ofdeveloping cancer (e.g., lung cancer, breast cancer, colon cancer, andprostate cancer), age-related diseases (e.g., cardiovascular disease,cerebrovascular disease, or Alzheimer's disease), tobacco-relateddiseases (e.g., emphysema, aortic aneurysms, esophageal cancer, orsquamous cell cancer of the head and neck), or other diseases anddisorders relating to or caused by cellular senescence by proceduresestablished in the art of diagnostic imaging. An agent labeled with aradionuclide metal, such as technetium-99 m, may be administered to amammal (e.g., a human) by intravenous injection in a pharmaceuticallyacceptable solution such as isotonic saline, or by other methodsdescribed herein. The amount of a labeled agent of the invention (e.g.,peptides, polypeptides, proteins, small molecules, antibodies, orantibody fragments that target senescent cells) appropriate foradministration is dependent upon the distribution profile of the chosenconjugate in the sense that a rapidly cleared agent may be administeredat higher doses than one that clears less rapidly. Unit doses acceptablefor imaging tissues that contain senescent cells are in the range of,e.g., 5-40 mCi for a 70 kg individual. In vivo distribution andlocalization can be tracked by standard techniques described herein atan appropriate time subsequent to administration; typically between 30minutes and 180 minutes depending upon the rate of accumulation at thetarget site with respect to the rate of clearance at non-target tissue.

Therapeutic or Cytotoxic Agents Coupled to Agents of the Invention

Any of the agents of the invention (e.g., peptides, polypeptides,proteins, small molecules, antibodies, or antibody fragments that targetsenescent cells) can be coupled to any known cytotoxic or therapeuticmoiety. Examples include, e.g., antineoplastic agents such as: Acivicin;Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin;Aldesleukin; Altretamine; Ambomycin; A. metantrone Acetate;Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase;Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa;Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin;Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Camptothecin; Caracemide; Carbetimer;Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin;Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine;Combretestatin A-4; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; DACA (N-[2-(Dimethyl-amino)ethyl]acridine-4-carboxamide);Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Dolasatins; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene;Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate;Eflornithine Hydrochloride; Ellipticine; Elsamitrucin; Enloplatin;Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole;Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium;Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate;Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine;Fludarabine Phosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone;Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198;Homocamptothecin; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b;Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; PeploycinSulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Rhizoxin; Rhizoxin D; Riboprine; Rogletimide; Safingol;Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium;Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin;Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur;Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine;Tomudex; TOP53; Topotecan Hydrochloride; Toremifene Citrate; TrestoloneAcetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate;Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa;Vapreotide; Verteporfin; Vinblastine; Vinblastine Sulfate; Vincristine;Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate;Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate;Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin;Zinostatin; Zorubicin Hydrochloride; 2-Chlorodeoxyadenosine; 2′Deoxyformycin; 9-aminocamptothecin; raltitrexed;N-propargyl-5,8-dideazafolic acid;2chloro-2′-arabino-fluoro-2′-deoxyadenosine; 2-chloro-2′-deoxyadenosine;anisomycin; trichostatin A; hPRL-G129R; CEP-751; linomide; sulfurmustard; nitrogen mustard (mechlor ethamine); cyclophosphamide;melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-Nnitrosourea(MNU); N, N′-Bis(2-chloroethyl)-N-nitrosourea (BCNU);N-(2-chloroethyl)-N′cyclohexyl-N-nitrosourea (CCNU);N-(2-chloroethyl)-N′-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU);N-(2-chloroethyl)-N′-(diethyl)ethylphosphonate-N-nitrosourea(fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide;temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; Cisplatin;Carboplatin; Ormaplatin; Oxaliplatin; CI-973; DWA 2114R; JM216; JM335;Bis(platinum); tomudex; azacitidine; cytarabine; gemcitabine;6-Mercaptopurine; 6-Thioguanine; Hypoxanthine; teniposide 9-aminocamptothecin; Topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin;darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D);amsacrine; pyrazoloacridine; all-trans retinol;14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-Hydroxyphenyl)retinamide; 13-cis retinoic acid; 3-Methyl TTNEB; 9-cis retinoic acid;fludarabine (2-F-ara-AMP); or 2-chlorodeoxyadenosine (2-Cda).

Other therapeutic compounds include, but are not limited to, 20-pi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, prostaticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid;bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;bisnafide; bistratene A; bizelesin; breflate; bleomycin A2; bleomycinB2; bropirimine; budotitane; buthionine sulfoximine; calcipotriol;calphostin C; camptothecin derivatives (e.g., 10-hydroxy-camptothecin);canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; 2′deoxycoformycin(DCF); deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; discodermolide;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflomithine; elemene; emitefur; epirubicin; epothilones (A, R═H; B,R=Me); epithilones; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide; etoposide4′-phosphate (etopofos); exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide;homoharringtonine (HHT); hypericin; ibandronic acid; idarubicin;idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones;imiquimod; immunostimulant peptides; insulin-like growth factor-1receptor inhibitor; interferon agonists; interferons; interleukins;iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor, leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maytansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; rnerbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; ifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mithracin;mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxinfibroblast growth factor-saporin; mitoxantrone; mofarotene;molgramostim; monoclonal antibody, human chorionic gonadotrophin;monophosphoryl lipid A+myobacterium cell wall sk; mopidarnol; multipledrug resistance gene inhibitor, multiple tumor suppressor 1-basedtherapy; mustard anticancer agent; mycaperoxide B; mycobacterial cellwall extract; myriaporone; N-acetyldinaline; N-substituted benzamides;nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin;nartograstim; nedaplatin; nemorubicin; neridronic acid; neutralendopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxideantioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone;oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oralcytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin;paclitaxel analogues; paclitaxel derivatives; palauamine;palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin;pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium;pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol;phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil;pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetinB; plasminogen activator inhibitor; platinum complex; platinumcompounds; platinum-triamine complex; podophyllotoxin; porfimer sodium;porfiromycin; propyl bis-acridone; prostaglandin J2; proteasomeinhibitors; protein A-based immune modulator; protein kinase Cinhibitor; protein kinase C inhibitors, microalgal; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors;purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethyleneconjugate; raf antagonists; raltitrexed; ramosetron; ras farnesylprotein transferase inhibitors; ras inhibitors; ras-GAP inhibitor;retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;roquinimex; rubiginone B 1; ruboxyl; safingol; saintopin; SarCNU;sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescencederived inhibitor 1; sense oligonucleotides; signal transductioninhibitors; signal transduction modulators; single chain antigen bindingprotein; sizofiran; sobuzoxane; sodium borocaptate; sodiumphenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stem cell inhibitor; stem-cell division inhibitors;stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactiveintestinal peptide antagonist; suradista; suramin; swainsonine;synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide;tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium;telomerase inhibitors; temoporfin; temozolomide; teniposide;tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide;thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin;thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone;tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan;topsentin; toremifene; totipotent stem cell factor; translationinhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate;triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growthinhibitory factor; urokinase receptor antagonists; vapreotide; variolinB; vector system, erythrocyte gene therapy; velaresol; veramine;verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

One or more of the agents of the invention (e.g., peptides,polypeptides, proteins, small molecules, antibodies, or antibodyfragments that target senescent cells) can also be coupled to a lyticpeptide. Such lytic peptides induce cell death and include, but are notlimited to, streptolysin O; stoichactis toxin; phallolysin;staphylococcus alpha toxin; holothurin A; digitonin; melittin;lysolecithin; cardiotoxin; and cerebratulus A toxin (Kem et al., J.Biol. Chem. 253(16):5752-5757, 1978). Agents of the invention can alsobe coupled to a synthetic peptide that shares some sequence homology orchemical characteristics with any of the naturally occurring peptidelysins; such characteristics include, but are not limited to, linearity,positive charge, amphipathicity, and formation of alpha-helicalstructures in a hydrophobic environment (Leuschner et al., Biology ofReproduction 73:860-865, 2005). Agents of the invention can also becoupled to an agent that induces complement-mediated cell lysis such as,for example, the immunoglobulin F_(c) subunit. Agents of the inventioncan also coupled to any member of the phospholipase family of enzymes(including phospholipase A, phospholipase B, phospholipase C, orphospholipase D) or to a catalytically-active subunit thereof.

Agents of the invention (e.g., peptides, polypeptides, proteins, smallmolecules, or antibody fragments that target senescent cells) can alsobe coupled to a radioactive agent, including, but not limited to:Fibrinogen ¹²⁵I; Fludeoxyglucose ¹⁸F; Fluorodopa ¹⁸F; Insulin ¹²⁵I;Insulin ¹²⁵I; Iobenguane ¹²³I; Iodipamide Sodium ¹³¹I; Iodoantipyrine¹³¹I; Iodocholesterol ¹³¹I; Iodohippurate Sodium ¹²³I; IodohippurateSodium ¹²⁵I; Iodohippurate Sodium ¹³¹I; Iodopyracet ¹²⁵I; Iodopyracet¹³¹I; Iofetamine Hydrochloride ¹²³I; Iomethin ¹²⁵I; Iomethin ¹³¹I;Iothalamate Sodium ¹²⁵I; Iothalamate Sodium ¹³¹I; tyrosine ¹³¹I;Liothyronine ¹²⁵I; Liothyronine ¹³¹I; Merisoprol Acetate ¹⁹⁷Hg;Merisoprol Acetate ²⁰³Hg; Merisoprol ¹⁹⁷Hg; Selenomethionine ⁷⁵Se;Technetium ^(99m)Tc Antimony Trisulfide Colloid; Technetium ^(99m)TcBicisate; Technetium ^(99m)Tc Disofenin; Technetium ^(99m)Tc Etidronate;Technetium ^(99m)Tc Exametazime; Technetium ^(99m)Tc Furifosmin;Technetium ^(99m)Tc Gluceptate; Technetium ^(99m)Tc Lidofenin;Technetium ^(99m)Tc Mebrofenin; Technetium ^(99m)Tc Medronate;Technetium ^(99m)Tc Medronate Disodium; Technetium ^(99m)Tc Mertiatide;Technetium ^(99m)Tc Oxidronate; Technetium ^(99m)Tc Pentetate;Technetium ^(99m)Tc Pentetate Calcium Trisodium; Technetium ^(99m)TcSestamibi; Technetium ^(99m)Tc Siboroxime; Technetium ^(99m)Tc;Succimer; Technetium ^(99m)Tc Sulfur Colloid; Technetium ^(99m)TcTeboroxime; Technetium ^(99m)Tc Tetrofosmin; Technetium ^(99m)TcTiatide; Thyroxine ¹²⁵I; Thyroxine ¹³¹I; Tolpovidone ¹³¹I; Triolein¹²⁵I; or Triolein ¹³¹I.

Therapeutic or cytotoxic agents may further include, for example,anti-cancer Supplementary Potentiating Agents, including, but notlimited to: Tricyclic anti-depressant drugs (e.g., imipramine,desipramine, amitryptyline, clomipramine, trimipramine, doxepin,nortriptyline, protriptyline, amoxapine, and maprotiline); non-tricyclicanti-depressant drugs (e.g., sertraline, trazodone, and citalopram);Ca²⁺ antagonists (e.g., verapamil, nifedipine, nitrendipine, andcaroverine); Calmodulin inhibitors (e.g., prenylamine,trifluoroperazine, and clomipramine); Amphotericin B; Triparanol analogs(e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine);antihypertensive drugs (e.g., reserpine); Thiol depleters (e.g.,buthionine and sulfoximine) and Multiple Drug Resistance reducing agentssuch as Cremaphor EL.

The agents of the invention (e.g., peptides, polypeptides, proteins,small molecules, antibodies, or antibody fragments that target senescentcells) can also be administered with cytokines, such as granulocytecolony stimulating factor, or anticancer agents used in anti-cancercocktails including, e.g., those shown with their MTDs in parentheses:gemcitabine (1000 mg/m²); methotrexate (15 gm/m² i.v.+leuco. <500 mg/m²i.v. w/o leuco); 5-FU (500 mg/m²/day×5 days); FUDR (100 mg/kg×5 in mice,0.6 mg/kg/day in human i.a.); FdUMP; Hydroxyurea (35 mg/kg/d in man);Docetaxel (60-100 mg/m²); discodermolide; epothilones; vincristine (1.4mg/m²); vinblastine (escalating: 3.3-11.1 mg/m², or rarely to 18.5mg/m²); vinorelbine (30 mg/m²/wk); meta-pac; irinotecan (50-150 mg/m²,1×/wk depending on patient response); SN-38 (−100 times more potent thanIrinotecan); 10-OH campto; topotecan (1.5 mg/m²/day in humans, 1×ivLDOmice-75 mg/m²); etoposide (100 mg/m² in man); adriamycin;flavopiridol; Cis-Pt (100 mg/m² in man); carbo-Pt (360 mg/m² in man);bleomycin (20 mg/m2); mitomycin C (20 mg/m²); mithramycin (30 sug/kg);capecitabine (2.5 g/m² orally); cytarabine (100 mg/m²/day);2-Cl-2′deoxyadenosine; Fludarabine-P04 (25 mg/m²/day, ×5 days);mitoxantrone (12-14 mg/m²); mitozolomide (>400 mg/m²); Pentostatin; orTomudex.

Any of the agents of the invention (e.g., peptides, polypeptides,proteins, small molecules, antibodies, or antibody fragments that targetsenescent cells) can be coupled to an antimetabolic agent. Antimetabolicagents include, but are not limited to, the following compounds andtheir derivatives: azathioprine, cladribine, cytarabine, dacarbazine,fludarabine phosphate, fluorouracil, gencitabine chlorhydrate,mercaptopurine, methotrexate, mitobronitol, mitotane, proguanilchlorohydrate, pyrimethamine, raltitrexed, trimetrexate glucuronate,urethane, vinblastine sulfate, vincristine sulfate, etc. In otherembodiments, the agents of the invention can be coupled to a folicacid-type antimetabolite, a class of agents that includes, for example,methotrexate, proguanil chlorhydrate, pyrimethanime, trimethoprime, ortrimetrexate glucuronate, or derivatives of these compounds.

In another embodiment, any of the agents of the invention (e.g.,peptides, polypeptides, proteins, small molecules, antibodies, orantibody fragments that target senescent cells) can be coupled to amember of the anthracycline family of neoplastic agents, including butnot limited to aclarubicine chlorhydrate, daunorubicine chlorhydrate,doxorubicine chlorhydrate, epirubicine chlorhydrate, idarubicinechlorhydrate, pirarubicine, or zorubicine chlorhydrate; a camptothecin,or its derivatives or related compounds, such as 10,11methylenedioxycamptothecin; or a member of the maytansinoid family ofcompounds, which includes a variety of structurally-related compounds,e.g., ansamitocin P3, maytansine, 2′-N-demethylmaytanbutine, andmaytanbicyclinol.

Any of the agents of the invention (e.g., peptides, polypeptides,proteins, small molecules, antibodies, or antibody fragments that targetsenescent cells) can be modified or labeled to facilitate diagnostic ortherapeutic uses. Detectable labels such as a radioactive, fluorescent,heavy metal, or other agents may be bound to any of the agents of theinvention. Single, dual, or multiple labeling of an agent may beadvantageous. For example, dual labeling with radioactive iodination ofone or more residues combined with the additional coupling of, forexample, 9Y via a chelating group to amine-containing side or reactivegroups, would allow combination labeling. This may be useful forspecialized diagnostic needs such as identification of widely dispersedsmall neoplastic cell masses.

Agents of the invention (e.g., peptides, polypeptides, proteins, smallmolecules, antibodies, or antibody fragments that target senescentcells), or analogs thereof, can also be modified, for example, byhalogenation of the tyrosine residues of the peptide component. Halogensinclude fluorine, chlorine, bromine, iodine, and astatine. Suchhalogenated agents may be detectably labeled, e.g., if the halogen is aradioisotope, such as, for example, ¹⁸F, ⁷⁵Br, ⁷⁷Br, ¹²²I, ¹²³I, ¹²⁴I,¹²⁵I, ¹²⁹I, ¹³¹I, or ²¹¹At. Halogenated agents of the invention containa halogen covalently bound to at least one amino acid, and preferably toD-Tyr residues in each agent molecule. Other suitable detectablemodifications include binding of other compounds (e.g., a fluorochromesuch as fluorescein) to a lysine residue of the agent of the invention,or analog, particularly an agent or analog having a linker includinglysines.

Radioisotopes for radiolabeling any of the agents of the invention(e.g., peptides, polypeptides, proteins, small molecules, antibodies, orantibody fragments that target senescent cells) include any radioisotopethat can be covalently bound to a residue of the peptide component ofthe agent of the invention or analog thereof. The radioisotopes can beselected from radioisotopes that emit either beta or gamma radiation, oralternatively, any of the agents of the invention can be modified tocontain chelating groups that, for example, can be covalently bonded tolysine residue(s) of the analog. The chelating groups can then bemodified to contain any of a variety of radioisotopes, such as gallium,indium, technetium, ytterbium, rhenium, or thallium (e.g., ¹²⁵I, ⁶⁷Ga,¹¹¹In, ^(99m)Tc, ¹⁶⁹Yb, ¹⁸⁶Re).

When one or more of the agents of the invention (e.g., peptides,polypeptides, proteins, small molecules, antibodies, or antibodyfragments that target senescent cells) is modified by attachment of aradioisotope, preferable radioisotopes are those having a radioactivehalf-life corresponding to, or longer than, the biological half-life ofthe agent used. More preferably, the radioisotope is a radioisotope of ahalogen atom (e.g. a radioisotope of fluorine, chlorine, bromine,iodine, and astatine), even more preferably ⁷⁵Br, ⁷⁷Br, ⁷⁶Br, ¹²²I,¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, or ²¹¹At.

Agents of the invention (e.g., peptides, polypeptides, proteins, smallmolecules, antibodies, or antibody fragments that target senescentcells) coupled to radioactive metals are useful in radiographic imagingor radiotherapy. Preferred radioisotopes also include ^(99m)Tc, ⁵¹Cr,⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ¹⁶⁸Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁵⁶Ho, ¹⁶⁵Dy, ⁶⁴Cu,⁹⁷R, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, and ²¹⁴Bi. Thechoice of metal can be determined based on the desired therapeutic ordiagnostic application.

The agents of the invention (e.g., peptides, polypeptides, proteins,small molecules, antibodies, or antibody fragments that target senescentcells), when coupled to a metal component, are useful as diagnosticand/or therapeutic agents. A detectable label may be a metal ion fromheavy elements or rare earth ions, such as Gd³⁺, Fe³⁺, Mn³⁺, or Cr²⁺.Conjugates that include paramagnetic or superparamagnetic metals areuseful as diagnostic agents in MRI imaging applications. Paramagneticmetals that can be coupled to the agents of the invention include, butare not limited to, chromium (III), manganese (II), iron (II), iron(III), cobalt (II), nickel (II), copper (II), praseodymium (III),neodymium (III), samarium (III), gadolinium (III), terbium (III),dysprosium (II), holmium (III), erbium (III), and ytterbium (III).Preferably, the polymer has a relaxtivity of at least 10, 12, 15, or 20mM⁻¹ sec⁻¹ Z⁻¹, wherein Z is the concentration of paramagnetic metal.

Chelating groups may be used to indirectly couple detectable labels orother molecules to an agents of the invention (e.g., a peptide,polypeptide, protein, small molecule, antibody, or antibody fragment).Chelating groups can link agents of the invention with radiolabels, suchas a bifunctional stable chelator, or can be linked to one or moreterminal or internal amino acid reactive groups. Conjugates can belinked via an isothiocyanate β-Ala or appropriate non α-amino acidlinker which prevents Edman degradation. Examples of chelators known inthe art include, for example, the ininocarboxylic andpolyaminopolycarboxylic reactive groups, ininocarboxylic andpolyaminopolycarboxylic reactive groups, diethylenetriaminepentaaceticacid (DTPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTA).

An agent of the invention (e.g., peptides, polypeptides, proteins, smallmolecules, antibodies, or antibody fragments that target senescentcells) can also be coupled directly to a cytotoxic or therapeutic agentusing known chemical methods, or the two moieties can be coupled via anindirect linkage, such as through a chelating group. For example, theagent can be attached to a chelating group that is attached to thecytotoxic or therapeutic agent. Chelating groups include, but are notlimited to, ininocarboxylic and polyaminopolycarboxylic reactive groups,diethylenetriaminepentaacetic acid (DTPA), and1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Forgeneral methods, see, e.g., Liu et al., Bioconjugate Chem. 12(4):653,2001; Cheng el al., WO 89/12631; Kieffer et al., WO 93/12112; Albert etal., U.S. Pat. No. 5,753,627; and WO 91/01144 (each of which are herebyincorporated by reference).

When coupled to a therapeutic or cytotoxic agent, specific targeting bythe agents of the invention (e.g., peptides, polypeptides, proteins,small molecules, antibodies, or antibody fragments that target senescentcells) allows selective destruction of senescent cells. For example, theagents of the invention can be used to target and destroy senescentcells of the lung, breast, prostate, and colon in order to prevent,stabilize, inhibit the progression of, or treat cancers originating inthese organs. Also, for example, the agents of the invention can be usedto target and destroy senescent cells of the vasculature, brain, liver,kidney, heart, lung, prostate, colon, nasopharynx, oropharynx, larynx,bronchus, and skin in order to prevent, stabilize, inhibit theprogression of, or treat age-related diseases or tobacco-relateddiseases or conditions relating to these organs. Any of the agents ofthe invention (e.g., peptides, polypeptides, proteins, small molecules,antibodies, or antibody fragments that target senescent cells) can beadministered to a mammalian subject, such as a human, directly or incombination with any pharmaceutically acceptable carrier or salt knownin the art. Pharmaceutically acceptable salts may include non-toxic acidaddition salts or metal complexes that are commonly used in thepharmaceutical industry. Examples of acid addition salts include organicacids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic,succinic, benzoic, palmitic, suberic, salicylic, tartaric,methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like;polymeric acids such as tannic acid, carboxymethyl cellulose, or thelike; and inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid phosphoric acid, or the like. Metal complexes includezinc, iron, and the like. One exemplary pharmaceutically acceptablecarrier is physiological saline. Other physiologically acceptablecarriers and their formulations are known to one skilled in the art anddescribed, for example, in Remington's Pharmaceutical Sciences, (18^(th)edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.

Agents of the Invention for Use in Cellular Therapy Applications

One or more agents of the invention (e.g., peptides, polypeptides,proteins, small molecules, antibodies, or antibody fragments that targetsenescent cells) can be used to improve the efficacy of a cellulartherapy provided to a patient (e.g., a mammal, such as a human) in needthereof by depleting (i.e., killing) one or more, all, or substantiallyall (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99% or more) senescent cells in the transferreddonor cells, tissue, or organ prior to, concurrent with, or followingadministration of the cellular therapy. Alternatively, a patient (e.g.,a mammal, such as a human) may be treated with one or more agents of theinvention prior to, concurrent with, or following administration of thecellular therapy. Cellular therapies include, without limitation, theautologous, allogeneic, syngeneic, or xenogeneic transplantation orengraftment of cells (e.g., blood, pancreatic islet cells, and stemcells (e.g., hematopoietic stem cells (HSC), umbilical cord blood stemcells (see, e.g., US Patent Application Publication 2002/0164794),pluripotent stem cells (e.g., hox11-expressing pluripotent stem cellsdescribed in US Patent Application Publication 2005/0158288),multipotent stem cells, and totipotent stem cells), tissues (e.g., skinand adipose tissue), or organs (e.g., kidney, liver, heart, and lung) toa patient (e.g., a mammal, such as a human) in need thereof. Cellulartherapies (e.g., cell (e.g., stem cells), tissue, or organtransplantation or engraftment) are currently being developed for a widerange of therapeutic indications for degenerative and pathologicdiseases, such as osteoarthritis (see, e.g., U.S. Patent ApplicationPublication No. 2007/0264238), ischemia and cardiac tissue damage, suchas that caused by myocardial infarction (see, e.g., Dzau et al., U.S.Patent Application Publication No. 2007/0259425), type I and type IIdiabetes (see, e.g., Uchida et al., U.S. Patent Application PublicationNo. 2007/0212732), renal dysfunction and multi-organ failure (see, e.g.,Westenfelder, U.S. Patent Application Publication No. 2007/0178071), andneurodegenerative diseases (see, e.g., Kim et al., U.S. PatentApplication Publication No. 2007/0054399). The preceding U.S. patentapplication Publications are hereby incorporated by reference in theirentirety.

Administration and Dosage

Pharmaceutical formulations of a therapeutically effective amount of anagent of the invention (e.g., peptides, polypeptides, proteins, smallmolecules, antibodies, or antibody fragments that target senescentcells), or pharmaceutically acceptable salt-thereof, can be administeredorally, parenterally (e.g., intramuscular, intraperitoneal, intravenous,or subcutaneous injection, inhalation, intradermally, optical drops, orimplant), nasally, vaginally, rectally, sublingually, or topically. Thepharmaceutical formulation can include the agent of the invention inadmixture with a pharmaceutically acceptable carrier adapted for theroute of administration.

Methods well known in the art for making pharmaceutical formulations canbe found, for example, in Remington's Pharmaceutical Sciences (18thedition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.Compositions intended for oral use may be prepared in solid or liquidforms according to any method known to the art for the manufacture ofpharmaceutical compositions. The compositions may optionally containsweetening, flavoring, coloring, perfuming, and/or preserving agents inorder to provide a more palatable preparation. Solid dosage forms fororal administration include capsules, tablets, pills, powders, andgranules. In such solid forms, the active compound is admixed with atleast one inert pharmaceutically acceptable carrier or excipient. Thesemay include, for example, inert diluents, such as calcium carbonate,sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodiumphosphate, or kaolin. Binding agents, buffering agents, and/orlubricating agents (e.g., magnesium stearate) may also be used. Tabletsand pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and soft gelatincapsules. These forms contain inert diluents commonly used in the art,such as water or an oil medium. Besides such inert diluents,compositions can also include adjuvants, such as wetting agents,emulsifying agents, and suspending agents.

Formulations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, or emulsions. Examples of suitablevehicles include propylene glycol, polyethylene glycol, vegetable oils,gelatin, hydrogenated naphthalenes, and injectable organic esters, suchas ethyl oleate. Such formulations may also contain adjuvants, such aspreserving, wetting, emulsifying, and dispersing agents. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for the agents of the invention include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes.

Liquid formulations can be sterilized by, for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilizing agentsinto the compositions, or by irradiating or heating the compositions.Alternatively, they can also be manufactured in the form of sterile,solid compositions which can be dissolved in sterile water or some othersterile injectable medium immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to active substances,excipients such as coca butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients known in the art. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops or spray, or as a gel.

The amount of active ingredient in the compositions of the invention canbe varied. One skilled in the art will appreciate that the exactindividual dosages may be adjusted somewhat depending upon a variety offactors, including the agent being administered, the time ofadministration, the route of administration, the nature of theformulation, the rate of excretion, the nature of the subject'sconditions, and the age, weight, health, and gender of the patient. Inaddition, the severity of the condition targeted by an agent of theinvention will also have an impact on the dosage level. Generally,dosage levels of an agent of the invention of between 0.1 μg/kg to 100mg/kg of body weight are administered daily as a single dose or dividedinto multiple doses. Preferably, the general dosage range is between 250μg/kg to 5.0 mg/kg of body weight per day. Wide variations in the neededdosage are to be expected in view of the differing efficiencies of thevarious routes of administration. For instance, oral administrationgenerally would be expected to require higher dosage levels thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, which are well known in the art. In general, the precisetherapeutically effective dosage can be determined by the attendingphysician in consideration of the above-identified factors.

An agent of the invention (e.g., a peptide, polypeptide, protein, smallmolecule, antibody, or antibody fragment that targets senescent cells)can be administered in a sustained release composition, such as thosedescribed in, for example, U.S. Pat. No. 5,672,659 and U.S. Pat. No.5,595,760. The use of immediate or sustained release compositionsdepends on the type of condition being treated. If the conditionconsists of an acute or over-acute disorder, a treatment with animmediate release form will be preferred over a prolonged releasecomposition. Alternatively, for preventative or long-term treatments, asustained released composition will generally be preferred.

An agent of the invention (e.g., a peptide, polypeptide, protein, smallmolecule, antibody, or antibody fragment that targets senescent cells)can be prepared in any suitable manner. The agent may be isolated fromnaturally-occurring sources, recombinantly produced, or producedsynthetically, identified from a library of small molecules, or producedby a combination of these methods. The synthesis of short peptides iswell known in the art. See e.g., Stewart et al., Solid Phase PeptideSynthesis (Pierce Chemical Co., 2d ed., 1984). A peptide portion of anyof the agents of the invention can be synthesized according to standardpeptide synthesis methods known in the art.

The present invention is illustrated by the following examples, whichare in no way intended to be limiting of the invention.

EXAMPLES Example 1 Discovery of Agents that Bind Senescent Cells PhageSelection Technique

Normal skin cell line CCD-1070Sk was obtained from American Type CultureCollection (Bethesda, Md.). The cells were grown in Eagle's MinimalEssential medium with Earle's BSS, 2 mM L-glutamine, 1.0 mM Sodiumpyruvate, 0.1 mM nonessential amino acids and 1.5 g/L sodium bicarbonatesupplemented with 10% fetal bovine serum.

Cells were sub-cultured every 4-5 days till they became senescent. Cellsenescence was confirmed by senescence-associated beta-galactosidasestaining.

An M13 phage peptide library, Ph.D.-12, was obtained from New EnglandBioLabs (Beverly, Mass.), which displays random 12-mer peptides.

For the selection step for round 1, an aliquot (10 μL) of the Ph.D.-12complete phage library was incubated with 5×10⁵ cells of senescentfibroblasts in 1 mL PBS/0.5% BSA for ˜3.5 hours at room temperature withslow shaking on Lab-Quake. At the end of the incubation, the cells werepelleted in a microcentrifuge at 1500 RPM for 2 minutes and thesupernatant removed. Cells were washed with PBS/1.0% BSA/0.5% Tween(wash buffer) for a total of 4 washes using fresh tubes between washes.Phage that bound to the target cells were eluted with 200 μL of 0.2 Mglycine (pH 2.2) for 8 minutes then neutralized with 30 μL of 1 MTris-HCl (pH 9.0). The number of phage bound was determined and theremaining eluate was amplified.

From the amplified eluate from Round 1, an aliquot (2×10¹¹ phage) wasused for subtraction panning against normal skin fibroblasts. The phagewere incubated with 2×10⁶ cells of normal skin fibroblast (CC D 1070 sk)at room temperature for 60 minutes at room temperature with slow shakingin PBS with 0.5% BSA. At the end of incubation, the cells were pelletedin a microcentrifuge at 1500 RPM for 2 minutes and the supernatantrecovered. The supernatant was used to resuspend another 2×10⁶subtraction cells. This subtraction step was repeated 3 times for eachround.

After the final subtraction step in each round, the recovered phagesupernatant was used to suspend 5×10⁵ selection cells. The bound phagewere recovered and amplified as described above. The process wasrepeated for a total of 5 rounds of selection. After the fifth round ofselection, phage were titered and 20 well-separated plaques were picked,amplified, and sequenced.

Results:

Three consensus sequences were obtained from the phage display selectionprocedure. These correspond to the SEQ ID NOS:1-3. SEQ ID NO:1represented 9/20 clones, SEQ ID NO:2 represented 6/20 clones, and SEQ IDNO:3 represented 2/20 clones.

Example 2 Human Study to Test the Prognostic Use of a Senescent CellBinding Agent Labeled with a Radioisotope; Using a Senescent CellDetecting Radiotracer to Predict Cancer Risk

This study is designed to show that noninvasive, in vivo imaging ofsenescent cell content can be used to predict cancer risk in smokers.Six-hundred subjects will be enrolled. Eighty percent of these subjectswill be smokers; twenty percent will be nonsmokers. Subjects less than18 years of age, subjects with a history of cancer, and pregnantsubjects will be excluded. The anticipated mean age of study subjects is55 years. Each study subject will undergo scintigraphic imaging using aradio-labeled peptide of the invention (SEQ ID NO:1) as theradiopharmaceutical. The radiopharmaceutical will be prepared byreacting peptide-gly-gly-gly-ser-DTPA with 111-In chloride. Each studysubject will receive an intravenously administered dose of 5 mCi of111-In labeled peptide. Anterior and posterior whole body planar imagesof the subjects will be obtained at 24 and 48 hours followingadministration of the radio-labeled peptide. SPECT imaging of the chestwill also be obtained at 24 and 48 hours following administration of theradio-labeled peptide. Scintigraphic images will be acquired on aSPECT/CT camera using a medium energy collimator. Two radiologists willread the SPECT/CT study of each subject. Regions of interest will bedrawn around each lung and the mediastinum. The activity within eachregion will be determined for each subject. Subjects will be followedfor two years and monitored for the incidence of lung cancer.Kaplan-Meier curves will be drawn for the study population based uponcancer free survival. An optimal threshold of activity within theregions of interest will be determined such as to divide subjects thatdevelop cancer from subjects who don't; a Kaplan-Meier curve will bedrawn for the set of subjects with activity levels above the thresholdand a Kaplan-Meier curve will be drawn for subjects with activity levelsbelow the threshold level. We expect to observe development of lungcancer in approximately twenty subjects. Activity in pulmonary regionsof interest will be significantly higher in smokers than nonsmokers.Smokers who develop lung cancer will have significantly higher activityin pulmonary regions of interest than smokers who do not develop lungcancer. Cancer-free survival curves will be significantly different forsubjects with a pulmonary region of interest activity level above thethreshold value than for subjects with activities below the thresholdvalue.

Example 3 Using Senescent Cell Binding Agents Coupled to CytotoxicAgents to Eliminate Senescent Cells Effect on Subsequent Development ofCancer

The purpose of this study is to show that elimination of some senescentcells from an organism using the agents of the invention (e.g.,peptides, polypeptides, proteins, small molecules, antibodies, orantibody fragments that target senescent cells) will reduce the risk ofsubsequently developing cancer. For example, a senescent cell bindingpeptide (SEQ ID NO:1) will be conjugated to a cytotoxic peptide havingthe sequence KFAKFAKKFAKFAKKFAKFAK (SEQ ID NO:4; Leuschner and Hansel,Biology of Reproduction 73:860-865) via an amino acid linker sequence(e.g., a linker sequence selected from GGGC (SEQ ID NO:9), GGGS (SEQ IDNO:10), and GG) at the C terminus of the senescent cell binding peptide.Study subjects will consist of 60 BALB/c mice, mean age 6 mo whichincludes 30 experimental animals and 30 controls. Experimental animalswill receive an intravenously administered dose of 0.2 mg/Kg of bodyweight of senescent cell binding peptide conjugate once every threemonths for one year. Control animals will receive an equal dose of a 37amino acid control peptide. Kaplan-Meier curves representing tumor freesurvival will be constructed for each group of mice. Approximately 30%of control mice will develop tumors by the end of the study period. Thestudy is expected to show significantly longer tumor free survival inthe experimental group compared to the control group.

Example 4 Reduction of Senescent Cell Content in Diabetic Mice byTreatment with a Senescent Cell Cytotoxic Agent

Diabetes is induced in female CD-1 mice, 5-7 weeks old and 25-35 g inbody weight, by intraperitoneal injection of 200 mg/Kg body weight ofstreptozotocin dissolved in sodium citrate saline buffer (pH 4.5). Tailvein blood glucose will be measured 5 days after injection to ensureinduction of diabetes. Diabetic mice will be maintained at constanttemperature (23° C.) with 12 hour light and 12 hour dark cycles for 16weeks following confirmation of diabetes. Seven diabetic mice willreceive a weekly tail vein injection of a senescent cell cytotoxic agent(SenL; SEQ ID NO:6; GVYHFAPLTPTPGGGSKFAKFAKKFAKFAK; 300 μg/dose),comprising a senescent cell binding sequence linked to a lytic peptidesequence during the 16 weeks. Seven control animals will receive anequivalent volume TV injection of normal saline. At the end of 16 weeks,all animals will be sacrificed. Tissue cross sections will be preparedfrom aorta, lung, liver, and heart from snap frozen tissue. Tissuesamples will be stained for SA-β-gal activity using the method ofCampisi et al. Percentage of SA-β-gal positive cells will be determinedfor each tissue sample from each animal by counting 1000 cells in eachof four random microscopic fields for each tissue sample. A two-tailedI-test will be used to evaluate the loss of senescent cells in thetissues of diabetic mice treated with senescent cell cytotoxic agentrelative to the loss of senescent cells in the tissues of diabeticcontrol mice.

Example 5 Enhancement of Stem Cell Treatments by Pre-Treatment withSenescent Cell Binding Agents Coupled to Cytotoxic Agents: Effects onSubsequent Stem Cell Engraftment

The following experiment can be used to show that exogenouslyadministered stem cells engraft at higher rates into damaged tissue ifthe treated organism undergoes pretreatment with a senescent cellcytotoxic agent of the invention (e.g., a peptide agent) to reduce thecontent of senescent cells in the damaged tissue compartment. Removal ofsenescent cells increases the engraftment of stem cells into damagedtissue.

Balb-C mice will undergo left anterior descending (LAD) artery ligationfor 60 minutes to induce myocardial infarction. The mice will bepre-anesthetized in an isoflurane inhalation chamber and receive an i.p.injection of sodium pentobarbital (25 mg/kg). The animals will beintubated and ventilated for the duration of the procedure. The LADartery will be identified following left lateral thoracotomy andpericardectomy. Ligation will be performed on the proximal 2 mm portionof the LAD using a 9-0 ethilon stitch. Mice will be maintained at 23 OCwith 12 hour light and 12 hour dark cycles for 6 days. Seven survivingmice will be used as experimental animals and seven will be used ascontrol animals. Experimental mice will receive tail vein injections ofa senescent cell cytotoxic agent conjugated to a lytic peptide sequence(SEQ ID NO:8; GVYHFAPLTPTPGGKFAKFAKKFAKFAK; 300 μg/dose) every secondday for six days.

Murine hematopoietic stem cells (HSC) will be obtained from StemCellTechnologies Inc. Cells will be transfected to express enhanced greenfluorescent protein (EGFP). Plasmid pEF-1a-EGFP, containing an EGFP geneunder the control of human EF1, a promoter, and a neomycin-resistancecassette, will be constructed as follows: (1) the promoter region ofpEGFP-N3 (Clontech, Palo Alto, Calif.) will be removed by cutting outthe AseI-NheI DNA fragment and joining the blunt-ended termini, and (2)human EF1, a promoter from pEF-BOS (a fragment of HindIII and EcoRI DNA)will be inserted into the HindI1-EcoRI site of the plasmid. Murine HSCwill be transfected with pEF-1a-EGFP by electroporation and selected inthe presence of G418. A single clone that brightly expresses EGFP willbe chosen and used for the experiments. The clone will be adapted tofeeder-free conditions and maintained on gelatin-coated dishes inDulbecco's Modified Eagle's Media supplemented with 15% fetal calfserum, 2 mM sodium pyruvate, 2 mM L-glutamine, 1× nonessential aminoacids, 1,000 units of 0.1 mM 2-mercaptoethanol per mL, along with 100units of streptomycin and 100 μg of penicillin per mL. Cells will becollected after trypsinization with EDTA and placed in aliquots of themedium described above for mouse tail vein injection 1 hour later.

HSC (10⁶) will be injected via tail vein into each experimental andcontrol mouse. Ten days later, each animal will be sacrificed. Heartswill be excised and fixed in 2% paraformaldehyde in phosphate-bufferedsolution (PBS) for 2 hours and cryoprotected in 30% sucrose overnight.Tissue will be embedded in optimum cutting temperature medium andsectioned at 5 μm on a cryostat. Serial sections will be stained withhematoxylin and eosin (H&E). Tissue will be examined with a fluorescentmicroscope. Percentage of GFP positive cells will be determined for eachcardiac tissue sample from each animal by counting 1000 cells in each offour random microscopic fields for each tissue sample. A two-tailedt-test will be used to evaluate the hypothesis that exogenouslyadministered HSC engraft at a higher rate into mice treated withsenescent cell cytotoxic agent relative to untreated controls. Specialattention will be paid to cardiac tissue in the LAD territory (anteriorwall) of each heart.

Example 6 In vitro Validation of the Use of Senescent Cell BindingAgents as Agents to Deliver Molecular Cargo to the Cytoplasm ofSenescent Cells Cytotoxicity of Senescent Cell Binding PeptideConjugated to a Lytic Peptide Sequence

Cytotoxic peptide SenL (i.e., SEQ ID NO:6) was synthesized byconjugating a senescent cell binding agent (SEQ ID NO:1) to a lyticpeptide sequence (KFAKFAKKFAKFAK; SEQ ID NO:4) via a 4 residue linker(GGGS; SEQ ID NO:10) and tested for differential cytotoxic activity insenescent fibroblasts, prostate epithelial cells, and non-senescentfibroblasts. Senescent fibroblasts exhibited dose-dependent cell deathat a significantly higher rate than either non-senescent fibroblasts orprostate epithelial cells (FIG. 1). The effect of SenL on cellproliferation was also assessed using the Cell Proliferation Assay WST-1(Roche, Mannheim, Germany). As shown in FIG. 2, no change in cellproliferation was seen in any of the three cell types in response totreatment with SenL. The proliferation assay uses WST-1 as a reagent;the reaction is catalyzed by mitochondrial dehydrogenases. Senescentcells have higher mitochondrial mass than their non-senescentcounterparts (Lee et al., J. Biomed. Sci. 9:517-26, 2002); consequently,baseline WST assay values are higher for senescent cells. The occurrenceof cell death in response to treatment with SenL and absence of changein proliferation rate indicate that SenL causes cell death innon-proliferating cells, e.g. senescent cells.

It is worth noting that each cultured cell population contains a mixtureof senescent and non-senescent cells at all population doublings, butthat the relative proportion of senescent cells within the populationincreases stochastically with each population doubling (Martin-Ruiz etal., J. Biol. Chem. 279(17):17826-33, 2004). Thus, the “senescent cells”used in this cell killing experiment are predicted to contain asubpopulation of non-senescent cells. Likewise, a fraction of the“non-senescent cells” used in this experiment are predicted to besenescent. Therefore, the observed difference in cell killing betweenthe two populations is reduced by the impure composition of eachpopulation with regard to senescence. This explains why somecytotoxicity is observed in the “non-senescent” fibroblast population.It also explains why the prostate epithelial cells (RWPE-1), which arenot predicted to have any senescent cells due to immortalization throughHPV-18 transduction, show no cell death at all.

In Vitro Cytotoxicity: Conjugation to Ricin-A

A senescent cell binding agent (i.e., SEQ ID NO:1) was conjugated to thericin A subunit via a 4 peptide linker (GGGC; SEQ ID NO:9) to produceSenR (SEQ ID NO:7). Senescent fibroblasts, non-senescent fibroblasts,and prostate epithelial cells were then incubated with the peptide-ricinA conjugate (SenR). Increased cell death was observed in the case ofsenescent cells treated with SenR than in the other cell types (FIG. 3).For example, an approximately 3 fold increase in cell death was measuredin senescent fibroblast when compared to non-senescent fibroblast whentreated with 50 μM of SenR.

Binding of Peptide to Senescent Cells Versus Non-Senescent Cells

In the absence of unlabeled peptide, radio-labeled SenC bound toprostate epithelium, normal fibroblasts, and senescent cells at anaverage of 0.06%, 0.08%, and 0.32% of added dose, respectively,demonstrating that SenC binds to senescent cells at a higher rate thanto the other cell types (P=0.001). In the presence of 20 μM unlabeledSenC, labeled SenC bound to prostate epithelium, normal fibroblasts, andsenescent cells at an average of 0.06%, 0.09%, and 0.25% of added dose,respectively. There was no significant difference between binding ratesin the presence or absence of unlabeled SenC in the case of prostateepithelium and normal fibroblasts (P=0.5 and 0.4 respectively),indicating that the binding to these cell types is nonspecific. LabeledSenC did bind at significantly different rates to senescent cells in theabsence vs. presence of unlabeled SenC (P=0.04), indicative of specificbinding (see FIG. 4).

Example 7 Isolation and Identification of Senescent Cell-SpecificAntigens

Fibroblasts (CCD-1070Sk) were cultured as outlined above and dividedinto three groups: replicatively senescent, stress induced prematurelysenescent, and non-senescent. Cell surface proteins of each group ofcells were conjugated to biotin using the Pierce Cell Surface ProteinIsolation Kit (Thermo Scientific, product number 89881) following theinstructions of the manufacturer. Membrane proteins were captured usingneutravidin following membrane dissolution and sent for 2D gelelectrophoresis. Spots from each gel were analyzed to look fordifferences in protein expression among the three groups of fibroblasts.Protein spots occurring in the gels corresponding to senescent(replicative or stress-induced) cells but not in gels corresponding tonon-senescent fibroblasts were identified as senescence-specificantigens and identified using MALDI-TOF mass spectrometry.

Mutant beta-actin (SEQ ID NO:11; GI: 28336) and ACTB protein (SEQ IDNO:12; GI: 15277503) were identified as cell-surface, senescencespecific antigens occurring in both replicatively senescent andstress-induced prematurely senescent cells. Drug resistance-relatedprotein LRP (SEQ ID NO:13; GI: 1097308) and major vault protein (SEQ IDNO:14; GI: 19913410) were identified as senescence-specific surfaceproteins in the replicatively senescent cells only. Senescence specificantigens that were identified in stress induced prematurely senescentcells included thyroid hormone binding protein precursor (SEQ ID NO:15;GI: 339647); unnamed protein product (SEQ ID NO:20; GI: 35655); prolyl4-hydroxylase, beta subunit precursor (SEQ ID NO:16; GI: 20070125);chain A, human protein disulfide isomerase (SEQ ID NO:17; GI:159162689); electron-transfer-flavoprotein, beta polypeptide (SEQ IDNO:18; GI: 4503609); unnamed protein product (SEQ ID NO:21; GI:158257194); unnamed protein product (SEQ ID NO:22; GI: 158259937); ATPsynthase, H transporting, mitochondrial F1 complex, alpha subunitprecursor (SEQ ID NO:19; GI: 4757810), and cathepsin B (SEQ ID NO:23).

Methods Induction of Replicative Senescence

CCD-1070Sk (fibroblasts) was cultured as described herein. Cells werecultured until they underwent 68 population doublings at which time theydisplayed typical senescent morphology and underwent minimal furthercell growth in response to mitogens.

Isolation of Cell Surface Proteins

Cell surface proteins were extracted from three groups of cells(replicatively senescent fibroblasts, stress-induced prematurelysenescent fibroblasts, and non-senescent fibroblasts). Each group ofcells contained 10⁹ cells. Cell surface proteins were isolated using thePierce Cell Surface Protein Isolation Kit (Thermo Scientific, productnumber 89881), following the instructions of the manufacturer. Proteinisolates were analyzed using spectrophotometry which showed absorbancesat 280 nm of 1.25, 1.375, and 1.347 for replicatively senescent cells,stress induced prematurely senescent cells, and non-senescent cellsrespectively. Total volume of each protein isolate was 500 μL.

Identification of Cell Surface Proteins

Cell surface protein isolates were sent for analysis by the proteomicscore at the University of Massachusetts Medical School (Worcester,Mass.). The analysis was carried out by performing a buffer exchange foreach protein isolate sample followed by 2D gel electrophoresis. Each gelwas compared to find protein spots that occurred in the gelscorresponding to the senescent (replicative or stress-induced) cells butnot in the gels corresponding to the non-senescent cells. Protein spotsthat occurred in the senescent cell samples but not in the non-senescentsamples were digested and sent for mass spectrometry analysis foridentification.

Example 8 Immunostaining for Cathepsin B Expression on the Surface ofSenescent Cells

Senescent fibroblasts and non-senescent fibroblasts were grown on coverslips and immunostained for cell surface expression of cathepsin B.Images appear in FIGS. 5A and 5B, which show surface staining forcathepsin B on the senescent cells but not on their non-senescentcounterparts.

Fibroblasts (CCD 1070Sk) were grown in culture as detailed herein.Replicatively senescent cells were acquired by growing cells for 50population doublings followed by plating on cover slips. Non-senescentcells were acquired by plating mid passage fibroblasts on cover slips.Cells on cover slips were allowed to attach overnight. Cells were fixedwith methanol and washed. Rabbit polyclonal antibody to cathepsin B wasdiluted 1:100 in PBS with 0.2% BSA. Cells were incubated with primaryantibody to cathepsin B for one hour at room temperature followed bywashing three times with cold PBS. Secondary antibody (goat anti rabbitIgG conjugated to FITC) was diluted 1:100 in PBS with 0.2% BSA and usedto incubate cells for 30 minutes at room temperature. Cells were washedthree times with cold PBS.

Example 9 Materials and Methods Cell Culture

All cells were obtained from American Type Culture Collection (Manassas,Va.). Each cell culture was grown at 37° C. in 5% CO₂. CCD-1070Sk(fibroblasts) were grown in minimum essential medium (Eagle) with 2 mML-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodiumbicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodiumpyruvate, and supplemented with 10% fetal bovine serum. RWPE-1(non-cancerous prostate epithelial cells) were grown inkeratinocyte-serum free medium supplemented with 5 ng/mL humanrecombinant EGF and 0.05 mg/mL bovine pituitary extract.

Chemical Induction of Cellular Senescence

CCD-1070Sk (fibroblasts) was cultured as described above to 70%confluence. Cells were then treated with 200 μM hydrogen peroxide (i.e.,H₂O₂) for 2 hours. Media was then replaced with fresh media and cellswere allowed to grow for 3 days. Cells were then harvested bytrypsinization, split 1:3, and again grown to 70% confluence. Cells werethen retreated with 200 μM hydrogen peroxide. Lower passage numberfibroblasts frequently required two treatments with hydrogen peroxide,but higher passage fibroblasts occasionally required only a singletreatment.

Peptide Synthesis

Peptides were synthesized using standard FMOC protected chemistry. Forin vitro cell cytotoxicity studies, peptide was synthesized with aC-terminal, cell lytic sequence according to the following:GVYHFAPLTPTPGGGS(KFAKFAK)₂ (SEQ: ID NO:6; SenL). The peptide sequenceGVYHFAPLTPTPGGGC (SEQ ID NO:5; SenC) was synthesized for subsequentconjugation to ricin-A subunit and for radio-labeling with99m-technetium.

Conjugation of FITC to Peptide

FITC was conjugated to the N terminus of SenC according to thefollowing: (1) A senescent cell binding peptide (SenC; SEQ ID NO:5) wasprepared at a concentration of 5 mM in 125 μL of 0.5 M NaHCO₃ buffer, pH9.5, and (2) FITC was added to the peptide solution at a 1:5 molar ratio(peptide:FITC) and diluted to a final volume of 200 μL. The solution wasincubated in the dark for 2 hours. Peptide-FITC conjugate was purifiedon a P4 column using PBS, pH 7.2 as an eluent.

Cell Internalization

Premature senescence of fibroblasts (CCD-1070Sk) was induced as outlinedabove. Non-senescent fibroblasts were grown in culture as detailedabove. Cells were harvested and added to collagen-coated coverslips andgrown in MEM plus 10% FBS overnight. Media was removed, and cells werewashed once with PBS. Minimal essential media without FBS was added tothe cells. A senescent cell binding peptide (SenC; SEQ ID NO:5)conjugated to FITC was added to cells on coverslips at a concentrationof 5 μM and incubated for 3 hours. Cells were than washed five timeswith PBS followed by fixation with 1:1 methanol/acetone for 10 minutesat −20° C. Coverslips were air dried and mounted in fluorescent mountingmedia with DAPI and visualized with an Olympus BX51 fluorescentmicroscope and DP70 digital camera with excitation and emissionwavelengths of 490 and 520 nm.

Cytotoxicity of Senescent Cell Binding Peptide Conjugated to a LyticPeptide Sequence

CCD-1070Sk and RWPE-1 were grown in culture as detailed above. Prematuresenescence of fibroblasts was chemically induced as described above.Cells were trypsinated and suspended in culture media containing 10%FBS. Cells were centrifuged at 1,000 rpm for 5 minutes. Supernatant wasremoved and cells were resuspended in 1 mL media without FBS. Cellsuspensions were diluted to 20,000 cells/75 μL. Twenty-five microlitersof appropriately-diluted agent solution (SenL; SEQ ID NO:6) was added tocell samples to give various concentrations of 0, 0.1, 0.5, 1.0, 2.5,5.0 or 10 μM. Each sample was prepared in triplicate. Cell suspensionswere transferred to a 96 well plate and incubated in the presence ofvarious concentrations of SenL for 2 hours at 37° C. Six samples of eachcell type contained no agent. The assay plate was removed from theincubator, and 2 μL of lysis solution (Tris 25 mM, pH 7.5, 0.5% tritonX-100) was added to three samples of each cell type without agent togenerate a positive control maximum LDH release. LDH release wasmeasured in each sample by adding 100 μL of CytoTox-ONE Reagent (RocheApplied Science) to each well and mixing on a plate shaker for 30seconds. Samples were incubated at 22° C. for 10 minutes. The reactionwas terminated by adding 50 μL of Stop Solution (per 100 μL ofCytoTox-ONE™ Reagent added) to each well. Fluorescence was measured ineach well using an excitation wavelength of 530 nm and an emissionwavelength of 620 nm (Cytofluor 4000). The CytoTox-ONE assay was shownto yield a quantity of fluorescent product that is linearly proportionalto the number of cells killed (correlation coefficient=0.99). Thepercentage of cells killed was calculated using the following formula:

${\% \mspace{14mu} {cytotoxicity}} = \frac{100\% \times \left( {P - C} \right)}{\left( {M - C} \right)}$

where P=LDH release in wells of peptide incubated cells; C=LDH releasein wells of cells not incubated with peptide; and M=LDH release in wellsincubated in lysis solution. The formula is based upon the assumptionsthat, in a linear relationship between CytoTox-ONE product developmentand number of cells killed, C is the y-intercept, and M is due to 100%cell killing.

Effect of SenL on Cell Proliferation

Cell proliferation was assayed using the Cell Proliferation ReagentWST-1 (Roche, Mannheim, Germany) by following the manufacturer'sinstructions.

In Vitro Cytotoxicity: Conjugation to Ricin-A

Peptide SenC (GVYHFAPLTPTPGGGC; SEQ ID NO:5) was conjugated to ricin Asubunit (Sigma-Aldrich) to form SenR. Ricin A was obtained frommanufacturer in solution. A buffer exchange was performed with 0.1 MPBS/20% glycerol. Ricin A was conjugated with NHS-PEO₄-maleimide crosslinker (Pierce, Rockford, Ill.) at a 1:10 molar ratio for 30 minutes atroom temperature. Derivatized ricin A was purified on a P4 column using0.1 M PBS/20% glycerol as an elution buffer. Derivatized ricin A wascombined with P12S at a 1:1 molar ratio and reacted for 2 hours at roomtemperature.

CCD-1070Sk and RWPE-1 were grown in culture as detailed above.Senescence was chemically-induced as described above. Cells weretrypsinized and suspended in culture media containing 10% FBS. Cellswere centrifuged at 1,000 rpm for 5 minutes. Supernatant was removed andcells were resuspended in 1 mL media without FBS. Cell suspensions werediluted to 20,000 cells/75 μL. Twenty-five microliters ofappropriately-diluted peptide-ricin A conjugate was added to each cellsample to give various concentrations. Each sample was prepared intriplicate. Cell suspensions were transferred to a 96 well plate andincubated in the presence of peptide-ricin A conjugate for 2 hours at37° C. Percentage of cells killed (FIG. 3) was determined as outlinedabove.

Radio-Labeling of Peptide Senescent Cell Binding Peptide

A senescent cell binding agent (SEQ ID NO:1) was conjugated at its Cterminus to the linker sequence GGGC (SEQ ID NO:9) by synthesizing bothas a single construct (i.e., SenC; SEQ ID NO:5). The purpose ofattaching GGGC was that it can be used to chelate reduced 99m-Tc forradio-labeling. A 2 μL aliquot of conjugated senescent cell bindingagent (3 M) was mixed with 40 μL of 0.25 M ammonium acetate, 15 μL oftartrate buffer pH 8.7, 4 μL of stannous chloride in 100 mM of sodiumtartrate, and 30 μL of 99m-Tc pertechnetate. The mixture was heated for25 minutes at 95° C. Quality control was performed with Sep-Pak and wasalways above 90% purity. A small aliquot was also injected on a Waters600 HPLC to check the radiological profile. Fractions were collected andread on a gamma counter (Perkin-Elmer Wallac Wizard 1470).

Cell Binding Assay

Binding of radio-labeled SenC (SEQ ID NO:5) to senescent andnon-senescent cells was tested by competition with unlabeled SenC.Peptide solutions were prepared to contain 0 or 20 μM of unlabeled SenC,15 nM radio-labeled SenC, and 0.2% BSA. Chemically-induced senescentfibroblasts, their non-senescent counterparts, and prostate epithelialcells were prepared as above, harvested, and centrifuged. The cellpellets were resuspended in fresh media without FBS, and cells werecounted. Peptide solution containing 0 or 20 μM unlabeled SenC andconstant concentrations of SenC and 10⁵ senescent cells were combined ina final volume of 200 μL of PBS in Eppendorf tubes and incubated for 4hours. Cells were pelleted by centrifuging at 2500 rpm for 2 minutes andwashed twice with PBS and 0.2% BSA. Pellets were suspended in 5 μL PBSand transferred to 12×75 mm tubes for counting radioactivity using agamma counter (Perkin-Elmer Wallac Wizard 1470).

OTHER EMBODIMENTS

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth.

1-54. (canceled)
 55. A method of treating a disease related to or causedby cellular senescence in a mammal comprising administering to themammal a pharmaceutical composition comprising: (a) a humanized antibodyor antibody fragment thereof, wherein the humanized antibody or antibodyfragment thereof specifically binds to a senescent cell; and (b) apharmaceutically acceptable carrier.
 56. The method of claim 55, whereinthe humanized antibody or antibody fragment thereof specifically bindsto a senescent cell-specific peptide, polypeptide, or glycoprotein onthe senescent cell.
 57. The method of claim 56, wherein the senescentcell-specific peptide, polypeptide, or glycoprotein is expressed on acell surface of the senescent cell.
 58. The method of claim 57, whereinthe senescent cell-specific peptide, polypeptide, or glycoprotein is asenescent cell-specific antigen comprising an amino acid sequence setforth in any one of SEQ ID NOS:11-23.
 59. The method of claim 55,wherein the humanized antibody of the antibody fragment thereof iscoupled to a therapeutic agent.
 60. The method of claim 59, wherein thetherapeutic agent is a cytotoxic agent.
 61. The method of claim 55,wherein the disease is a cancer, an age-related disease, or atobacco-related disease.
 62. The method of claim 61, wherein theage-related disease is selected from the group consisting ofcardiovascular disease, cerebrovascular disease, osteoarthritis, benignprostatic hypertrophy, and Alzheimer's disease.
 63. The method of claim61, wherein the tobacco-related disease is selected from the groupconsisting of emphysema, aortic aneurysms, esophageal cancer, peripheralvascular disease, cardiac diastolic dysfunction, aortic aneurysm, andsquamous cell cancer.
 64. A method for inducing apoptosis in a senescentcell in a mammal comprising administering to the mammal a pharmaceuticalcomposition comprising (a) a humanized antibody or antibody fragmentthereof, wherein the humanized antibody or antibody fragment thereofspecifically binds to the senescent cell; and (b) a pharmaceuticallyacceptable carrier.
 65. The method of claim 64, wherein the humanizedantibody or antibody fragment thereof specifically binds to a senescentcell-specific peptide, polypeptide, or glycoprotein.
 66. The method ofclaim 65, wherein the senescent cell-specific peptide, polypeptide, orglycoprotein is expressed on a cell surface of the senescent cell. 67.The method of claim 66, wherein the senescent cell-specific peptide,polypeptide, or glycoprotein is a senescent cell-specific antigencomprising an amino acid sequence set forth in any one of SEQ IDNOS:11-23.
 68. The method of claim 64, wherein the humanized antibody orthe antibody fragment that is administered to the mammal is coupled to atherapeutic agent.
 69. The method of claim 68, wherein the therapeuticagent is a cytotoxic agent.
 70. The method of claim 64, wherein thesenescent cell is a senescent lung cell, a senescent breast cell, asenescent colon cell, a senescent prostate cell, a senescent gastriccell, a senescent hepatic cell, a senescent ovarian cell, a senescentesophageal cell, a senescent bronchial epithelial or stromal cell, asenescent skin epithelial or stromal cell, a senescent glial cell, or asenescent vascular endothelial or stromal cell.
 71. The method of claim64, wherein the humanized antibody or antibody fragment thereofcomprises (a) amino acid sequence selected from any one of SEQ IDNOS:1-3 in a complementarity determining region; or (b) an amino acidsequence at least 90% identical to the amino acid sequence selected fromany one of SEQ ID NOS:1-3 in a complementarity determining region.
 72. Amethod for depleting senescent cells from donor cells, a donor tissue,or a donor organ, said method comprising (a) contacting the donor cells,the donor tissue, or the donor organ with a humanized antibody orantibody fragment thereof, wherein the humanized antibody or antibodyfragment thereof specifically binds to a senescent cell; and (b)administering the donor cells, the donor tissue, or the donor organ tothe mammal prior to, concurrent with, or subsequent to the step ofcontacting.
 73. The method of claim 72, wherein the humanized antibodyor antibody fragment thereof is administered to the mammal prior to,concurrent with, or subsequent to administration of the donor cells, thedonor tissue, or the donor organ to the mammal.
 74. The method of claim72, wherein the humanized antibody or antibody fragment thereofcontacting the donor cells, tissue, or organ is coupled to a therapeuticagent.
 75. A pharmaceutical composition comprising: (a) a humanizedantibody or antibody fragment thereof coupled to a therapeutic agent,wherein the humanized antibody or antibody fragment thereof specificallybinds to a senescent cell-specific peptide, polypeptide, orglycoprotein, and wherein the therapeutic agent is capable of killing asenescent cell; and (b) a pharmaceutically acceptable carrier.
 76. Thepharmaceutical composition of claim 75, wherein the humanized antibodyor antibody fragment thereof comprises (a) an amino acid sequenceselected from any one of SEQ ID NOS:1-3 in a complementarity determiningregion; or (b) an amino acid sequence at least 90% identical to theamino acid sequence selected from any one of SEQ ID NOS:1-3 in acomplementarity determining region.
 77. The pharmaceutical compositionof claim 75, wherein the senescent cell-specific peptide, polypeptide,or glycoprotein is a senescent cell-specific antigen comprising an aminoacid sequence set forth in any one of SEQ ID NOS:11-23,